How Pipette Tips Influence Assay Results

Home , Plate reader, Shaker (laboratory), Turbidostat

No. 44 – 2016

(BN 44) JANUARY 2016 PAGE 5

Fast, Precise, and Sterile Liquid Transfer in Cell Culture with Multipette® M4 /Repeater® M4

NATHALIE CHANDELIER, VINCENT DUFEY, MURIEL ART, EPPENDORF APPLICATION TECHNOLOGIES S.A., NAMUR, BELGIUM DIANA HÜBLER, EPPENDORF AG, HAMBURG, GERMANY

Abstract Materials and methods The most important factors in cell culture are sterility, accu- Instrument calibration racy, and precision. Positive displacement dispensers offer Systematic and random errors were determined by gravimetric the great advantage of complete aerosol prevention as the method in accordance with EN ISO 8655:2002 [1]. The volumes piston integrated in the tip of the positive displacement tested were 100 µL in each pipetting system. Workflow Solutions for Microbiology and Fermentation system hermetically seals the sample in the tip to prevent > contamination. In regard to accuracy and precision positive Cell counting displacement dispensers show equivalent results to those Cell number was determined by using the CASY® Cell Counter obtained with air-cushion pipettes. In contrast, reproducibil- and Analyser, model TT 150 µm (Roche®, Basel, Switzerland). ity of results will be affected when pipette controllers in Measurement was performed by suspending 100 µL of cell combination with serological pipets are used. suspension in 10 mL of CASY ton. Introduction Cell seeding and WST-1 colorimetric assay Animal cell culture has become a common laboratory tech- After cell counting, a cell suspension of 1.5 x 106 HEK 293 nique and is used for a large range of applications. The most cells/mL was prepared for cell seeding. Various quantities of important factors to consider are sterility in terms of con- cells were seeded into 24-well plates (Eppendorf). 40 µL of tamination prevention and accuracy, as well as precision. > Avoiding the Negative Effects of Leachables Water Soluble Tetrazolium salt (WST-1; Roche) were added Commonly, pipette controllers with serological pipets are per well. Cell culture plates were incubated for 3 h (37 °C,

used for the transfer of large volumes. Air-cushion pipettes 5 % CO2). The plates were mixed in a plate reader and read are used for all other parts of the application. But one of the at 450 nm (reference: 690 nm). greatest sources of contamination is aerosol formation dur- Results and discussion ing pipetting, therefore using a two phase filter tip is recommended by Eppendorf. Performance evaluation of the used pipetting systems An alternative to these systems are positive displacement Each instrument was used with its dedicated consumable dispensers, such as the Multipette M4 (U.S.: Repeater M4), (Multipette M4 /Repeater M4 with Combitips advanced® 1 mL, that prevent contamination of the instrument or cross-con- Eppendorf Research® plus pipette with ep Dualfilter T.I.P.S.® tamination via aerosols completely. This is ensured by a pis- 100 µL, Eppendorf Xplorer® pipette with ep Dualfilter T.I.P.S. > Automated NGS Sample Preparation ton that hermetically seals the sample in the tip of the posi- 1 mL, and Easypet® 3 with 1 mL Eppendorf Serological Pipets). tive displacement system. Easypet 3 is the least accurate and least reproducible system of all four systems (Fig. 1 and 2). Furthermore this system allows for accurate and precise pipetting results especially when working with liquids whose In contrast, with mean systematic and random errors below physical properties differ from those of water (e.g. DMSO, 0.6 %, air-cushion pipettes and Multipette M4/Repeater M4 cell culture medium). appear as the most reliable systems.

1 2 3.0 14.0 n = 3 n = 3 12.0

2.0 Application Notes 10.0 8.0 1.0 6.0 Random error [%]

Systematic error [%] 4.0 ® ® 0.0 2.0

-1.0 0.0 Liquid Transfer in Cell Culture with Multipette M4/Repeater M4 · Automated Bioreactor Research plus Xplorer Multipette M4/ Easypet Research plus Xplorer Multipette M4/ Easypet Repeater M4 Repeater M4 Instrument Instrument

Fig. 1 and 2: Systematic (Fig. 1) and random error (Fig.2) at 100 µL measured for the different pipetting systems used

Your local distributor: www.eppendorf.com/contact Sampling for Improved Microbial Strain Characterization · etc. Eppendorf AG · 22331 Hamburg · Germany · e-mail: [email protected] · www.eppendorf.com 2 EDITORIAL · DEAR READERS

Imprint

Editorial team Berrit Hoff (Editor-in-Chief), Axel Jahns, Jochen Müller-Ibeler, Natascha Weiß

Publisher Eppendorf AG, Barkhausenweg 1, 22339 Hamburg, Germany Telephone: (+49) 40-53801-636 Fax: (+49) 40-53801-840 E-mail: [email protected] Internet: www.eppendorf.com

We welcome all readers’ articles for this publication. However, no responsibility is accepted for unsolicited manuscripts.

Important note Dear Readers, The new products described may be launched at different times in various Quality, reliability, experience, and innovation are terms which users worldwide associate countries. Please contact your local with Eppendorf. Our excellent reputation is the result of 70 years of striving to offer the Eppendorf organization or distributor best solutions for handling precious samples in life sciences research. For example, for details. liquid handling systems that just get it right – precision, reliability, and reproducibility. Technical specifications subject to change. Such as Eppendorf pipettes and Eppendorf pipette tips – two perfectly matched system Errors and omissions excepted. components which provide you with the certainty of obtaining reliable, consistent assay results (pages 4 – 5 and Application Notes pages 1– 2). All rights reserved, including graphics and images. The responsible selection of even supposedly “routine products” such as tips and tubes © Copyright Eppendorf AG, January 2016. undoubtedly contributes to higher precision and reliability, as well as to increased con- Carbon neutrally printed in Germany. fidence in experimental data. For our consumables, we exclusively use carefully selected raw materials that are not recycled. During production, neither plasticizers, nor biocides or mold release agents are used, since especially these additives have been linked to “leaching” effects which can exert a strong negative impact on experiments (p. 8). If you are involved with microbial fermentation we have got news for you! The upcoming introduction of the new BioBLU® 3f Single-Use Vessel will expand our range of single- use bioreactors for the cultivation of bacteria, yeast, and fungi to a volume range from 65 mL to 3.75 L (pages 6 – 7). Additional workflow solutions for microbiology and fermentation are introduced on pages 10 –11. Starting with this issue, we will discontinue the traditional service fax. You can download the brochures for individual products denoted by reference numbers from the BioNews service site www.eppendorf.com/bn-service. Here you can also submit the solution to our new contest (p. 15). For personal feedback, praise, or criticism, you can always reach us by e-mail at [email protected]. We look forward to hearing from you!

Your BioNews team CONTENTS 3

4 9 11

IN THE SPOTLIGHT How “Fitting” Pipette Tips Interfere with Assay Results 4 – 5 STRAIGHT FROM THE LAB Consumables: Avoiding the Negative Effects of Leachables 8 Workflow Solutions for Microbiology and Fermentation 10 –11 Automated NGS Sample Preparation 12 INNOVATION BioBLU® 3f: Defining Single-Use Fermentation 6 – 7 CLOSE-UP Fill 96 Wells at once with epMotion® 96 9 NEWS / TIPS Extra Long, Extra Slim, Extra Safe – a New 5 mL L Tip! 5 Biological Shaker Performance Plans for Consistent, Reliable Operation 7 Good Analysis Makes Good Beer! 8 Liquid Handling Excellence Rewarded 9 Eppendorf App: Enter the Digital World of Eppendorf 12 Research Prizes: Winners Visit Eppendorf 13 For Additional Information, Please Consult the Operating Manual! 14 SERVICE Trademarks Information 14 Prize Competition 15

(BN 44) JANUARY 2016 PAGE 5 MURIEL ART, VINCENT DUFEY, IOAN GLIGOR, ULRIKE GAST, RONJA KUBASCH Fast, Precise, and Sterile Liquid Transfer in Cell Culture with Multipette® M4 /Repeater® M4 NATHALIE CHANDELIER, VINCENT DUFEY, MURIEL ART, EPPENDORF APPLICATION TECHNOLOGIES S.A., NAMUR, BELGIUM The Tip of the Iceberg: How Pipette Tips Influence Results. DIANA HÜBLER, EPPENDORF AG, HAMBURG, GERMANY 1 – 2

Abstract Materials and methods The most important factors in cell culture are sterility, accu- Instrument calibration racy, and precision. Positive displacement dispensers offer Part 1: Tip Fit Is Not All Users Should Look for Systematic and random errors were determined by gravimetric the great advantage of complete aerosol prevention as the method in accordance with EN ISO 8655:2002 [1]. The volumes piston integrated in the tip of the positive displacement tested were 100 µL in each pipetting system. system hermetically seals the sample in the tip to prevent contamination. In regard to accuracy and precision positive Cell counting displacement dispensers show equivalent results to those Cell number was determined by using the CASY® Cell Counter obtained with air-cushion pipettes. In contrast, reproducibil- and Analyser, model TT 150 µm (Roche®, Basel, Switzerland). ity of results will be affected when pipette controllers in Measurement was performed by suspending 100 µL of cell MATTHEW J. MAURER, JEFFREY M. SKERKER, ADAM P. ARKIN, WILLIAM MILLER, combination with serological pipets are used. suspension in 10 mL of CASY ton. Introduction Cell seeding and WST-1 colorimetric assay Animal cell culture has become a common laboratory tech- After cell counting, a cell suspension of 1.5 x 106 HEK 293 nique and is used for a large range of applications. The most MICHAEL BIKSACKY, CLAUDIA M. HUETHER-FRANKEN, KARL RIX cells/mL was prepared for cell seeding. Various quantities of important factors to consider are sterility in terms of con- cells were seeded into 24-well plates (Eppendorf). 40 µL of tamination prevention and accuracy, as well as precision. 3 – 4 Water Soluble Tetrazolium salt (WST-1; Roche) were added Commonly, pipette controllers with serological pipets are per well. Cell culture plates were incubated for 3 h (37 °C, used for the transfer of large volumes. Air-cushion pipettes 5 % CO2). The plates were mixed in a plate reader and read are used for all other parts of the application. But one of the at 450 nm (reference: 690 nm). Automated Bioreactor Sampling greatest sources of contamination is aerosol formation dur- Results and discussion ing pipetting, therefore using a two phase filter tip is recommended by Eppendorf. Performance evaluation of the used pipetting systems An alternative to these systems are positive displacement Each instrument was used with its dedicated consumable dispensers, such as the Multipette M4 (U.S.: Repeater M4), (Multipette M4 /Repeater M4 with Combitips advanced® 1 mL, that prevent contamination of the instrument or cross-con- Eppendorf Research® plus pipette with ep Dualfilter T.I.P.S.® tamination via aerosols completely. This is ensured by a pis- 100 µL, Eppendorf Xplorer® pipette with ep Dualfilter T.I.P.S. ton that hermetically seals the sample in the tip of the posi- 1 mL, and Easypet® 3 with 1 mL Eppendorf Serological Pipets). NATHALIE CHANDELIER, VINCENT DUFEY, MURIEL ART, DIANA HÜBLER tive displacement system. Easypet 3 is the least accurate and least reproducible system of all four systems (Fig. 1 and 2). Furthermore this system allows for accurate and precise pipetting results especially when working with liquids whose In contrast, with mean systematic and random errors below physical properties differ from those of water (e.g. DMSO, 0.6 %, air-cushion pipettes and Multipette M4/Repeater M4 Fast, Precise, and Sterile Liquid Transfer in Cell Culture cell culture medium). appear as the most reliable systems. 5 – 6

1 2 3.0 14.0 ® ® n = 3 n = 3 12.0 with Multipette M4/Repeater M4 2.0 10.0

8.0 1.0 6.0 Random error [%]

Systematic error [%] 4.0 0.0 STACEY WILLARD, MA SHA 2.0

-1.0 0.0 Research plus Xplorer Multipette M4/ Easypet Research plus Xplorer Multipette M4/ Easypet Repeater M4 Repeater M4 Instrument Instrument Solving the Aggregation Problem of HEK293 Cells Fig. 1 and 2: Systematic (Fig. 1) and random error (Fig.2) at 100 µL measured for the different pipetting systems used 7 – 8

Your local distributor: www.eppendorf.com/contact ™ Eppendorf AG · 22331 Hamburg · Germany · e-mail: [email protected] · www.eppendorf.com Using the New Brunswick S41i CO 2 Incubator Shaker 4 IN THE SPOTLIGHT · HOW “FITTING” PIPETTE TIPS INTERFERE WITH ASSAY RESULTS

BRIGITTE KLOSE, EPPENDORF AG How “Fitting” Pipette Tips Interfere with Assay Results

There is more to pipetting than the simple transfer of small and smallest amounts of liquid. The exact and coor- dinated interaction between pipette and pipette tip is of critical importance. Both components, each on its own as well as together as a system, must meet high quality standards in order to be able to deliver reliable, precise, and reproducible results. With this article, we want to sensitize you to the serious influence pipette tips have on the pipetting result.

When was the last time you were upset tips were awarded more consideration and Users can rely on the fact that each lot about non-reproducible results, for ex- selected on the basis of solid expertise. of the manufactured pipette tips will cor- ample in a real-time PCR? You checked respond exactly to this calibration result, Study examining pipette tips by all the variables in your experiment but i.e. no altered results will be obtained 15 manufacturers were still unable to find the cause? Dur- during pipetting. The system manufac- ing pipetting (the most frequent step in We have approached this topic by con- turer tightly coordinates his production any experimental process) you always ducting a broadly designed study of of pipettes and tips with respect to pro- used a pipette with a “fitting” tip – thus pipette tips by 15 global manufacturers duction tolerances and thus ensures that a system made up of two components, (see Application Notes pages 1– 2). Of the system operates within specifications the precision and accuracy of which you course, many more manufacturers exist; at all times. thought you could rely on during the whole however, the tips selected by us are those This is not the case for non-system workflow. But perhaps you could not? which in our experience are most fre- manufacturers; they do not have system quently found in laboratories, without Purchasing processes with far- specifications. If a pipette by one manu- discriminating between tips by “system reaching consequences facturer is used with a pipette tip by a providers” of pipettes and pipette tips different manufacturer, neither of them Great attention is paid to the purchase of and “non-system providers”, i.e. manu- will take responsibility for the precision a new pipette in the laboratory. Its tech- facturers of plastic labware only. and accuracy of the system. nology must be up-to-date, and handling Requirements of ISO 8655:2002 must be ergonomic. It should be robust and resistant to chemicals, and of course The international standard ISO 8655:2002 deliver accurate and precise results. The considers pipettes and pipette tips as a latter, however, can only be achieved with system and requires an extra calibration the right pipette tip. And this is where when using alternative pipette tips. Even contradictions frequently arise: While the if the pipette tips by manufacturer A “fit” pipette is chosen with great care, the se- the pipette by manufacturer B, this does lection of the pipette tip is often subject not speak to the precision and accuracy to drastically lower expectations (and of the pipetting result. budgets), assuming that they are just After testing two different volume sizes another consumable. of pipette tips by all 15 manufacturers Especially when purchasing products on the same pipettes, it became obvious: destined for daily routine, such as pipette the pipette tip significantly impacts the tips, many laboratories favor price over result. When using pipette and pipette quality. Unfortunately without knowledge tip by a system provider, the user can of the consequences. Many non-repro- safely assume that errors will not occur, The continuously high quality of the tip is a prerequisite for ducible experimental results – and there- since pipette and tip are calibrated and reproducible results. The image shows an epT.I.P.S.® pipette fore additional costs and expenditure of adjusted by the manufacturer in accor- tip with ergonomically optimized cone geometry, easily recognizable by the droplet-shaped relief elements near the time – could be avoided if suitable pipette dance with ISO 8655:2002. upper rim. HOW “FITTING” PIPETTE TIPS INTERFERE WITH ASSAY RESULTS · IN THE SPOTLIGHT 5

News Extra Long, Extra Slim, Extra Safe!

A new 175 mm long 5 mL “L” epT.I.P.S.® pipette tip complements Eppendorf’s portfolio of pipette tips. Compared to the 120 mm long 5 mL version already available, this new tip is not just an entire 55 mm longer, but it is also considerably slimmer. This results in decisive advantages for the user and improved safety during pipetting. You can now easily reach the bottom of a multitude of vessels, bottles, and flasks commonly used in the laboratory and, thus, reduce sample loss. The slim tip design facilitates pipetting without touching the vessel wall or bottle neck, thus helping to prevent cross-contamination of both vessel and sample. The clearly visible graduation of the tip allows visual volume control at all times.

The new epT.I.P.S. 5 mL L are available in ISO 8655:2002 recommends the use of pipette and pipette tip by the same manufacturer. epT.I.P.S. are optimally compatible with Eppendorf pipettes. purity grades Eppendorf Quality™ and Biopur® as well as ep Dualfilter T.I.P.S.® in In this case the user is forced to perform pipette tips. This may be one reason why PCR clean/Sterile. calibration and, if applicable, adjustment scientific publications rarely, if ever, list of the pipette with the other tips – every the complete name, manufacturer, and time a new lot is used. lot number of the tips used. Errors caused by the pipetting system cannot be traced, Tip design also plays a role and reproduction of the data by other During our studies we found out that the scientific groups is no longer possible. tip design, too, has a large impact on the Don’t let it get this far and read more on pipetting result. Those who pipet a lot are this topic in the Application Note “The often familiar with pipette tips by differ- Tip of the Iceberg: How Pipette Tips In- ent manufacturers and know that many fluence Results. Part 1: Tip Fit Is Not All different shapes of pipette tips exist. Users Should Look for”, on pages 1– 2 in Some are longer and slimmer than oth- the center section of this issue. ers. There is the “rocket shape”, as well as the regular conical pipette tip. Such different shapes influence the pipetting process. The longer the tip, with other- wise identical inner dimensions, the higher the air-cushion inside the tip. Form follows function! The new extra-long tip allows you to easily reach the bottom of Eppendorf Since pipettes are always adjusted to a Conical Tubes 15 mL (Fig.) and other standard labo- defined air-cushion inside the pipette, ratory vessels. The slim outline enables touch-free immersion into the vessel. a change in air-cushion, especially for larger nominal volumes, will impact More information at www.eppendorf.com/ accuracy. consumables Often the user is not aware of the consequences the final result of their work will Family Brochure: Eppendorf Liquid Handling Consumables • ® suffer as a result of a careless choice of Ref. no. 288 epT.I.P.S. 5 mL L • Ref. no. 286 6 INNOVATION · BIOBLU® 3F: DEFINING SINGLE-USE FERMENTATION

ULRIKE BECKEN, EPPENDORF AG, BIOPROCESS CENTER EUROPE, JUELICH, GERMANY KEVIN VOLL, EPPENDORF, INC., ENFIELD, USA BioBLU® 3f: Defining Single-Use Fermentation

Single-use technology provides several advantages for time- and cost-effective bioprocessing, like reduced risk of contamination, shortened time between runs, and lower capital investment. With the launch of the BioBLU® 3f vessel for microbial fermentation Eppendorf extends its portfolio of single-use bioreactors for the cultivation of bacteria, yeasts, and fungi. BioBLU f Single-Use Vessels now cover working volumes from 65 mL to 3.75 L, allowing greater flexibility in research and process development.

Today’s bioprocessing needs smart demands on bioreactor design and func- BioBLU 3f – the newest family member solutions tionality make the use of single-use tech- From cost-efficient process development nology for the cultivation of bacteria and Manufacturing of enzymes, production in small volumes towards large-scale man- fungi more challenging. With the BioBLU f of antibiotics or fabrication of food sup- ufacturing, scalability is key. Researchers Single-Use Vessel family Eppendorf sets plements … the list of goods produced have benefited from the BioBLU 0.3f and new standards in microbial fermentation. by microbial fermentation is long, and BioBLU 1f for culture sizes from 65 mL with new, innovative bioprocess solutions The extensive experience Eppendorf has to 1.25 L. With the upcoming launch of researchers are constantly pushing the in polymer technology facilitated the the BioBLU 3f Eppendorf now extends its limits. To outweigh the costs for raw development of single-use bioreactors portfolio towards higher culture volumes materials and labor, the optimal use of optimized for microbial applications. Their up to 3.75 L (Fig. 1). resources is more important than ever. rigid wall eliminates the risk of damage Industrial design ensures scalability and during installation. The single layer poly- Careful selection and optimization of simplifies technology transfer and pro- mer design of vessel body and head plate bacterial and fungal strains boost prod- cess development (Table 1). mitigates issues with leachables and uct yields. A thorough understanding of extractables. During injection molding critical process parameters paves the no additives such as softeners are used. way to consistent product quality. Conse- The raw material is free of animal com- quently, researchers need user-friendly ponents and is USP Class VI certified. fermentation systems which facilitate time-efficient process optimization and provide for easy scale-up towards larger production volumes. Defining single-use fermentation Traditionally, stirred-tank bioreactors are made of glass or stainless steel. However, single-use vessels provide significant advantages such as eliminating the risk of cross contamination and reduced time between runs thanks to the omission of laborious cleaning procedures. This allows the researcher a greater peace of mind and helps to increase productivity and reduce overall costs. Fig. 1: BioBLU 0.3f, 1f, and 3f. Expansion of mammalian cells in single- The new BioBLU 3f extends use tanks like the BioBLU c vessels is the Eppendorf portfolio of single-use vessels for micro- firmly established. However, the specific bial applications. BIOBLU® 3F: DEFINING SINGLE-USE FERMENTATION · INNOVATION 7

BioBLU 0.3f BioBLU 1f BioBLU 3f

Ratio Hi /Di 1.8 2.0 2.0 News

Ratio hVwmax /Di 1.2 1.4 1.5 Number of impellers 2 2 or 3 3 Biological Shaker

Ratio d/Di 0.4 0.4 0.4

Table 1: Scalability by industrial design: Dimensions of the BioBLU f Single-Use Vessels. Performance Plans Hi: inner vessel height; Di: inner vessel diameter; hVwmax: maximal liquid height; d: impeller diameter Biological shakers are required to operate 220 continuously for days, weeks, or even months. Consistent, reliable shaker 3 L glass vessel 200 operation with repeatable performance is BioBLU 3f vessel essential to ensure that cell growth and 180 proliferation continue under stable, predictable environmental conditions. Even

160 relatively small, short-term variations in shaker operation can significantly influence

140 culturing outcomes, with negative effects on yields and/or cell expression.

120 With our Performance Plans options we 600 will make sure that your instruments are OD 100 in good working condition by professional inspection and maintenance service. 80

Performance Plans feature: 20 >>A choice of preventive maintenance 0 programs covering cleaning, inspection, 0 1 2 3 4 5 6 7 8 9 10 11 12 13 and maintenance work Time [h] >>Validation and adjustment of operating Fig. 2: E. coli K12 growth curves in BioBLU 3f Single-Use Vessels and conventional glass vessels are highly comparable. parameters in accordance to Eppendorf Fermentation was performed using an Eppendorf BioFlo 320 control station. Cell growth was monitored by offline measurement of the optical density (OD600). specifications >>Installation Qualification certification As good as glass and DASGIP® Parallel Bioreactor Sys- >>Operational Qualification certification tems, BioFlo® 320, and New Brunswick™ >>Full documentation BioBLU f vessels combine the advantages BioFlo 115 and 310. Designed as drop-in of single-use with the reliable performance Your benefits: replacement for autoclavable bioreactors, of glass or stainless steel bioreactors. BioBLU f vessels can be installed quickly >>Avoid unexpected downtime and failure Industrial design makes the BioBLU f and operated without additional training in your processes vessels meet the high demands of mass of staff. >>Long lifetime of your instrument transfer and heat removal of microbial >>Maintain consistency and reproducibility fermentation. Proven stirred-tank design, Single-use now! of your results powerful overhead drives featuring Rush- BioBLU f vessels for microbial fermenta- >>Confirmation of instrument performance ton-type impellers for excellent mixing, tion have been designed combining know- within manufacturer’s specification and innovative baffles for cooling let bac- how in bioprocessing with expertise in teria grow as efficiently as in convention- For more information please visit polymer technology. Developed as an al glass vessels (Fig. 2). www.eppendorf.com/epServices easy-to-use replacement for conventional or local websites.* Switching to single-use technology is glass bioreactors they allow fermentation easy scientists to benefit from the advantages of single-use technology. BioBLU f fermentation vessels are compatible with the Eppendorf benchtop BioBLU® f • Ref. no. 279 *Performance Plans (also for other Eppendorf bioreactor systems, including DASbox® products) are available in selected countries only. 8 STRAIGHT FROM THE LAB · CONSUMABLES: AVOIDING THE NEGATIVE EFFECTS OF LEACHABLES

NILS GERKE, EPPENDORF AG Consumables: Avoiding the Negative Effects of Leachables

Many life sciences laboratories still un- Stand up to leachables with Eppendorf derestimate the influence of substances With our long-standing expertise in man- which may leach from plastic consum- Tip ufacturing high quality plastic consumables. These “leachables” are capable of ables, the entire process chain receives interfering with experiments to a large due consideration during development degree. Good Analysis and production of Eppendorf consum- Do not underestimate leaching effects ables. This translates to: Only carefully Makes Good Beer! selected raw materials are used, which Experimental design often focuses exclu- were not recycled. During production, The brewery industry employs a number sively on immediate reaction conditions neither plasticizers, nor biocides or of different photometric analysis methods such as the optimization of concentrations mold release agents are used, since es- used for quality control of the final prod- of reaction solutions, or temperature reg- pecially these additives have been linked uct, as well as for monitoring the entire ulation. General experimental conditions, to leaching effects which may negatively brewing process. The methods focus on which also include the plastic consum- influence your experiments. color, possible turbidities, and specific ables, are often neglected. compounds which may influence the taste Webinar “Good Plastics, Bad Plastics” However, leaching effects are capable of and stability of beer. causing considerable distortion to the If you would like to learn more about “Short Protocols” for beer analysis results of even standard laboratory this topic, we recommend the webinar methods such as photometric detection “Good Plastics, Bad Plastics” at Eppendorf offers five Short Protocols for of nucleic acids and proteins. www.eppendorf.com/webinar_gpbp. beer analysis, which may be downloaded at www.eppendorf.com/shortprotocols-beer. A simple test such as heating water in In addition, comprehensive information These protocols describe the processes reaction tubes, followed by a subsequent as well as recommendations on how to involved in common brewery methods, as absorbance scan, sufficed to detect UV- evaluate, minimize, and control the risk sourced from the Method Collection of absorbent leachables in consumables of leaching effects, are available at MEBAK® (Mitteleuropäische Brautechnische made by other manufacturers, which www.eppendorf.com/consumables. Analysenkommission; www.mebak.org), may subsequently lead to an overesti- and other sources. They encompass tests mate of DNA concentrations (Fig. 1). for determining color, bitterness, and other substances relevant to quality such as vicinal diketones, thiobarbituric acid, and

Absorbance scan of water following incubation for 30 min at 95 °C free amino nitrogen.

2.25 The experiments were carried out in an 2.00 0.10 Eppendorf BioSpectrometer®. The free

1.75 0.08 selection of wavelengths in a spectral range of 200 nm to 830 nm facilitates 1.50 0.06 method establishment on this instrument. 1.25 0.04

1.00 0.02 Extinction [OD]

Extinction [OD] 0.75 0.00 240 260 280 300 0.50 Wavelength [nm] Fig. 1: Leaching effects may cause erroneous DNA 0.25 measurements in 1.5 mL tubes made by manufactur- 0.00 ers V and A1. For further 190 210 230 250 270 290 310 330 350 370 390 information see Eppendorf Wavelength [nm] Application Note No. 235 which can be downloaded Eppendorf Manufacturer V Manufacturer A1 at www.eppendorf.com/ Eppendorf BioSpectrometer® • Ref. no. 242 applications. (BN 44) JANUARY 2016 PAGE 1

The Tip of the Iceberg: How Pipette Tips Influence Results. Part 1: Tip Fit Is Not All Users Should Look for

MURIEL ART, VINCENT DUFEY, IOAN GLIGOR, EPPENDORF APPLICATION TECHNOLOGIES S.A., NAMUR, BELGIUM ULRIKE GAST, RONJA KUBASCH, EPPENDORF AG, HAMBURG, GERMANY

Abstract order to achieve efficient tip fit. Other Calibration was performed using ana- problems often remain unrecognized lytical balance Model XP26PC (Mettler- The fact that a tip fits onto a pipette like decreased pipette accuracy when Toledo ®) at 100 % nominal volume and cone does not say anything about the using other tips than recommended by 10 % nominal volume. Two series of 10 performance of the pipetting system the pipette supplier. pipettings were performed. Systematic comprising the components “Pipette error and random error were determined and Tip”. We performed a study includ- The ISO 8655-2:2002 standard [1] de- for each series of 10 measurements and ing standard tips from 15 different fines pipette and tip as a system, which compared to specifications [1] and [2]. manufacturers in order to investigate requires extra calibration for the use of the tip-related influence on the pipet- other manufacturers’ tips. But why does For further information please refer to ting result. The study results showed this standard put so much focus on a [3]. a dramatic influence of the tip on the product that is to be discarded after Results and discussion pipetting accuracy. usage? While being perfectly within error limits The standard ISO 8655 regards pipette This series of articles answers this with Eppendorf tips, we found the system and tip to be a system. Our study results question. It shows the influence of tips “Pipette and Tip” to be out of specifica- emphasize the validity of this statement on the pipetting result explaining the tions when using other manufacturers’ and the need of calibration / pipette main tip-related impact factors. tips. adjustment if other tips than recom- Material and methods mended by pipette manufacturer are As shown in Fig. 1 and 2, the allowed sys- to be used. General material tematic error was exceeded with 4 manufacturers’ tips at a volume of 1,000 µL Introduction Eppendorf Xplorer® plus 50 –1,000 µL and with 5 manufacturers’ tips at 1 µL and 0.5 –10 µL were used. Racked 10 µL Within the scientific community, a ris- test volume. These tips with 1,000 µL and 1,000 µL standard tips of Eppendorf ing number of published experiments test volume exceeded not only the manu- and 14 other manufacturers have been cannot be reproduced by other groups. facturer specifications but also the wider tested. Exceptions: Manufacturer H did In general, plastics are not taken seri- limits for systematic error as stated by not offer racked 10 µL standard tips, ously leading to problems with analysis the ISO 8655:2002 standard [1]. The manufacturers K and N offered only results caused by e.g. leachables or in- random error was noticeably increased 1,250 µL tips for 1,000 µL pipettes. correct pipetting volumes. This may but stayed within allowed tolerances. lead to results not being reproducible if Calibration by gravimetric method When comparing the calibration results performed by other groups using other The performance of the system “Pipette with the outcome of dimensional mea- consumables. and Tip” was determined by calibration surements, it becomes clear that with Some problems with pipette tips are according to [1]. Environmental condi- 1,000 µL the biggest impact factor is obvious, e.g. the need to push tips tions were set according to requirements the air-cushion size: Those tips that pro- with force onto the pipette cone in [1]. duced error limits beyond the pipette

1 µL 10 µL 2 0.5

0.45

0.4 1.5 0.35

0.3

1 0.25

0.2

Random error [%] error Random [%] error Random 0.15 0.5 0.1

0.05

0 0 -4 -2 0 2 4 6 8 10 -1.5 -1 -0.5 0 0.5 1 1.5 Systematic error [%] Systematic error [%]

Fig. 1: Calibration results using 10 µL tips of different manufacturers. The red shaded area shows the span of the maximum permissible errors stated for the system “Pipette and Tip” by pipette supplier. All data points within the red shaded area are within the specifications.

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The Tip of the Iceberg: How Pipette Tips Influence Results. Part 1: Tip Fit Is Not All Users Should Look for

100 µL 1,000 µL 3.5 3.5

3 3

2.5 2.5

2 2

1.5 1.5

Random error [%] error Random 1 [%] error Random 1

0.5 0.5

0 0 -5 -4 -3 -2 -1 0 -5 -4 -3 -2 -1 0 Systematic error [%] Systematic error [%]

Fig. 2: Calibration results using 1,000 µL tips of different manufacturers. The red shaded area shows the span of the maximum permissible errors stated for the system “Pipette and Tip” by pipette supplier. All data points within the red shaded area are within the specifications. specifications were longer although on different pipettes. However, the fact Literature they had a similar inner diameter [3]. that a tip physically fits onto a pipette [1] DIN EN ISO 8655:2002, parts 1, 2, 6. cone does not say anything about the Piston-operated volumetric apparatus. Larger tips offer a larger air-cushion. system’s performance. If other manu- Beuth-Verlag, Berlin, Germany. Since air-cushion pipettes are adjusted facturers’ tips are to be used, the only [2] Eppendorf Xplorer® plus operating manual. to a certain air-cushion size, an increase user’s chance to learn about inaccurate www.eppendorf.com/manuals in air-cushion volume has a negative pipetting results is by calibration. [3] Application Note No. 354: The tip of the impact on the pipetting result, espe- iceberg: How pipette tips influence results. cially with nominal volume. This is supported by statements from www.eppendorf.com/applications the standard [1] which declares that The calibration results have been the conformity certificate does not found to be independent of the pipette apply if pipettes and tips of different manufacturer and were reconfirmed by manufacturers are used. Therafter the calibrating with another manufacturer’s user is required to calibrate first with pipette (data not shown). the tips recommended by the pipette In contrast to 1,000 µL tips, the impact manufacturer (“conformity testing” to of “air-cushion size” does not influence ensure the system is working correctly), the 10 µL tips to an important degree. and secondly to calibrate with other With this tip model, other influencing manufacturers’ tips [1]. factors such as geometry and quality of Conclusion tip orifice are more important. These influencing factors are discussed in That a tip physically fits onto a pipette more detail in [3] and our next publica- cone does not mean that the system tion of BioNews. performs within the manufacturer’s specification. We have shown for The results are conclusive that precau- 1,000 µL and 10 µL tips that the system’s tion is needed if tips with a design dif- performance can be heavily influenced fering from the tip recommended by by the tip. the pipette supplier are to be used. Elongated tips are an example. Within Manufacturer-wise pipettes are supplied the operating manual, Eppendorf ad- adjusted to a certain air-cushion size. vises the user to adjust the pipette if The tip design, however, directly influ- such tips are to be utilized. In the case ences the air-cushion size. Especially of manual pipettes, this can be done by with bigger volumes such as 1,000 µL, user adjustment. More conveniently, this happens to a degree affecting the with electronic Xplorer pipettes, this system’s accuracy. With small volumes, is performed by just choosing the tip other impacting factors come into play Readers’ service from the options menu. Most non-sys- which will be discussed in more detail Family Brochure: Eppendorf Liquid Handling Consumables • Ref. no. 288 tem tip providers inform about tip fit in the next edition of BioNews.

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Automated Bioreactor Sampling: Process Trigger Sampling for Enhancing Microbial Strain Characterization

MATTHEW J. MAURER1, JEFFREY M. SKERKER1,2,3, ADAM P. ARKIN1,2,3, WILLIAM MILLER4, MICHAEL BIKSACKY4, CLAUDIA M. HUETHER-FRANKEN5 AND KARL RIX5 1 UC BERKELEY ENERGY BIOSCIENCES INSTITUTE (EBI), BERKELEY, CA, USA; 2 UC BERKELEY DEPARTMENT OF BIOENGINEERING, BERKELEY, CA, USA; 3 PHYSICAL BIOSCIENCES DIVISION, LAWRENCE BERKELEY NATIONAL LABORATORY, BERKELEY, CA, USA; 4 FLOWNAMICS® ANALYTICAL INSTRUMENTS, INC., MADISON, WI, USA; 5 EPPENDORF AG, BIOPROCESS CENTER, JUELICH, GERMANY CORRESPONDENCE: [email protected]

Abstract Materials and methods between the DASGIP system and third- party analytical devices. The Quantitative Engineering of Industrial Eppendorf DASGIP® Parallel Bioreactor Yeast program at the Energy Biosciences Systems Seg-Flow® Automated On-line Sampling Institute (EBI) focuses on a systems-level System Eppendorf DASGIP Parallel Bioreactor understanding of bacterial and yeast Systems enable parallel experimenta- The patented Seg-Flow 4800 Automated metabolism, gene regulation, and stress tion intended to accelerate process On-line Sampling System (Seg-Flow response for elucidating principles to development and increase throughput. System) is a liquid and data management help rationally engineer bacteria and Using DASGIP Control software (now device designed to withdraw samples yeasts for improved biofuel production Eppendorf DASware® control 5), multiple from up to eight bioreactors and deliver from lignocellulosic sources [1]. bioreactor vessels are controlled by a them to up to four analytical instruments To optimize yeast strain characterization single computer, enabling the experi- and/or fraction collectors. and selection researchers have imple- menter to test multiple conditions side- The FlowWeb™ software platform, which mented automated processes, including by-side and to run multiple independent controls all the Seg-Flow System func- the use of an integrated parallel biore- experiments simultaneously [2]. The tions, provides seamless connectivity actor system and automated bioreactor Eppendorf software DASware analyze with various third-party analyzers for sampling system. enables bidirectional communication enabling real-time analysis of process parameters such as nutrients, metabo- lites, and various cell measurements. Upon completion of the analysis, the Seg-Flow System acquires and processes the analyzer data. The FlowWeb OPC software suite communicates the analyzer data into any OPC-enabled supervisory control and data acquisition (SCADA) system. Process trigger sampling The Seg-Flow System is capable of performing automated sampling and analysis during planned or unplanned process events in response to an exter- Fig. 1: Architecture for the Seg-Flow 4800 process trigger sampling function. The process event or “trigger” is user-defined nal SCADA or other bioprocess man- and is programmed in the bioreactor’s OPC-enabled SCADA or bioprocess management system, which remotely controls the Seg-Flow System. agement system such as the DASGIP Control/DASware software platform.

DASbox/ Seg-Flow System The process events used to activate, or Off-line HPLC DASGIP System with FlowFraction 400 trigger, the Seg-Flow System are user- defined. Examples of process events include pH or dissolved oxygen excur- sions, culture induction, feeding, or OPC Off-line other in-process control actions. The process events require OPC data tag configuration and must be programmed into the host SCADA/bioprocess management system. When the process DASGIP Controller FlowWeb Process event and DASware analyze (OPC Client) with OPC Server Sample analysis event is detected by the bioreactor station, the data trigger is communicated

Fig. 2: DASGIP/Seg-Flow Process Trigger Sampling System. OPC communication permits continuous bi-directional com- to the SCADA system to commence munication between the Seg-Flow Automated On-line Sampling System and DASware control. The Eppendorf DASGIP/ the remote activation of the Seg-Flow DASbox system detects the user-defined process event and remotely activates the Seg-Flow System to perform the process trigger sampling function. System and a sample is automatically

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Automated Bioreactor Sampling: Process Trigger Sampling for Enhancing Microbial Strain Characterization

Fig. 3: Process Trigger Sampling Data using DASware Plant Overview Function. Plot displays (A) time of Seg-Flow activation by the DASGIP controller (vessel 1 = green, vessel 2 = red); (B) time and duration of Seg-Flow sample collection (vessel 1 = orange, vessel 2 = blue); (C) time of sample deposition into vial and vial position (vessel 1 = magenta, vessel 2 = green); (D) culture density data from biomass probe (vessel 1 = purple, vessel 2 = blue) and (E) cumulative media addition (vessel 1 = black, vessel 2 = magenta). withdrawn from the bioreactor. Upon The DASGIP Control system activated aligning the remotely controlled sample completion of the sample collection or process media feed and removal from collection with the process event (Fig. 3). analysis, the data is communicated to the culture vessels in response to optical Conclusion the SCADA/bioprocess management density measurements, and user-defined system via OPC over the laboratory values of media feed volume addition Coupling the Eppendorf DASGIP Paral- network (Fig. 1). were used as the process trigger events lel Bioreactor and Seg‑Flow automated for the Seg-Flow sampling system. on-line sampling technologies enabled This unique remote control function al- EBI’s Microbial Characterization Facility lows “around-the-clock” monitoring When the desired values of media feed research staff to implement remote- and sampling of unique process events volume addition were reached, the pro- controlled, automated process trigger that could impact process productivity cess trigger start command was com- sampling as an integral part of its yeast and/or product quality. municated by the DASGIP Control sys- strain characterization activities. Incor- tem to the Seg‑Flow System (Fig. 3). Results and discussion porating such tools can significantly Upon activation, the Seg-Flow System reduce project timelines and increase Integrating the Seg-Flow and Eppendorf withdrew the programmed sample vol- the efficiency of the microbial strain DASGIP Parallel Bioreactor System ume from the bioreactor and delivered characterization and selection process. Using OPC communication, the Seg-Flow the sample to the FlowFraction™ 400 Literature automated on-line sampling system was fraction collector. The collected sample integrated with the Eppendorf DASGIP was stored in the fraction collector until [1] Energy Biosciences Institute. Biofuels Production Research Programs. Programs and Parallel Bioreactor System (Fig. 2). the sample was analyzed using an off- Projects. 24 March 2014. line HPLC or other analyzer. http://www.energybiosciencesinstitute.org/ Process event tags were configured research/biofuels#1. and programmed in the DASGIP Control Vessel-specific sample collection data [2] Knocke, C., Vogt, J. Biofuels – Challenges & software. The Eppendorf DASware included the beginning and end of the Chances: How Biofuel Development can Benefit analyze OPC client facilitated connectiv- Seg-Flow sample collection phase as from Advanced Process Technology. Eng. Life ity between the FlowWeb OPC server well as the sample collection vial position. Sci. 9-2 (2009): 96-99. and the DASGIP Control system. All data were date- and time-stamped This publication was presented at the “Recent in the FlowWeb software, communicated Advances in Fermentation Technology (RAFT Process trigger sampling 11)” meeting, Clearwater Beach, Florida, USA. to the DASGIP Control software using Two yeast cultures were cultivated over the FlowWeb OPC Server and recorded The complete version of this Application Note (No. 298) can be downloaded in PDF format at a 2.5 day duration using a continuous- in the DASGIP Control software. This www.eppendorf.com/applications. culture process. A turbidostat control sample collection data was synchro- loop was employed to maintain a pre- nized in real-time with the fermentation Readers’ Service scribed biomass concentration as mea- process information and the Seg-Flow DASGIP® Parallel Bioreactor Systems for microbial applications sured by an in-situ optical density probe. activation time (process trigger time), • Ref. no. 287

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Fast, Precise, and Sterile Liquid Transfer in Cell Culture with Multipette® M4 /Repeater® M4

NATHALIE CHANDELIER, VINCENT DUFEY, MURIEL ART, EPPENDORF APPLICATION TECHNOLOGIES S.A., NAMUR, BELGIUM DIANA HÜBLER, EPPENDORF AG, HAMBURG, GERMANY

Abstract Materials and methods The most important factors in cell culture are sterility, accu- Instrument calibration racy, and precision. Positive displacement dispensers offer Systematic and random errors were determined by gravimetric the great advantage of complete aerosol prevention as the method in accordance with EN ISO 8655:2002 [1]. The volumes piston integrated in the tip of the positive displacement tested were 100 µL in each pipetting system. system hermetically seals the sample in the tip to prevent contamination. In regard to accuracy and precision positive Cell counting displacement dispensers show equivalent results to those Cell number was determined by using the CASY® Cell Counter obtained with air-cushion pipettes. In contrast, reproducibil- and Analyser, model TT 150 µm (Roche®, Basle, Switzerland). ity of results will be affected when pipette controllers in Measurement was performed by suspending 100 µL of cell combination with serological pipets are used. suspension in 10 mL of CASY ton. Introduction Cell seeding and WST-1 colorimetric assay Animal cell culture has become a common laboratory tech- After cell counting, a cell suspension of 1.5 x 106 HEK 293 nique and is used for a large range of applications. The most cells/mL was prepared for cell seeding. Various quantities of important factors to consider are sterility in terms of con- cells were seeded into 24-well plates (Eppendorf). 40 µL of tamination prevention and accuracy, as well as precision. Water Soluble Tetrazolium salt (WST-1; Roche) were added Commonly, pipette controllers with serological pipets are per well. Cell culture plates were incubated for 3 h (37 °C, used for the transfer of large volumes. Air-cushion pipettes 5 % CO2). The plates were mixed in a plate reader and read are used for all other parts of the application. But one of the at 450 nm (reference: 690 nm). greatest sources of contamination is aerosol formation dur- Results and discussion ing pipetting, therefore using a two-phase filter tip is recommended by Eppendorf. Performance evaluation of the used pipetting systems An alternative to these systems are positive displacement Each instrument was used with its dedicated consumable dispensers, such as the Multipette M4 (U.S.: Repeater M4), (Multipette M4 /Repeater M4 with Combitips advanced® 1 mL, that prevent contamination of the instrument or cross-con- Eppendorf Research® plus pipette with ep Dualfilter T.I.P.S.® tamination via aerosols completely. This is ensured by a pis- 100 µL, Eppendorf Xplorer® pipette with ep Dualfilter T.I.P.S. ton that hermetically seals the sample in the tip of the posi- 1 mL, and Easypet® 3 with 1 mL Eppendorf Serological Pipets). tive displacement system. Easypet 3 is the least accurate and least reproducible system of all four systems (Fig. 1 and 2). Furthermore this system allows for accurate and precise pipetting results especially when working with liquids whose In contrast, with mean systematic and random errors below physical properties differ from those of water (e.g. DMSO, 0.6 %, air-cushion pipettes and Multipette M4/Repeater M4 cell culture medium). perform as the most reliable systems.

1 2 3.0 14.0 n = 3 n = 3 12.0

2.0 10.0

8.0 1.0 6.0 Random error [%] error Random

Systematic error [%] error Systematic 4.0 0.0

2.0

-1.0 0.0 Research plus Xplorer Multipette M4/ Easypet Research plus Xplorer Multipette M4/ Easypet Repeater M4 Repeater M4 Instrument Instrument

Fig. 1 and 2: Systematic (Fig. 1) and random error (Fig. 2) at 100 µL measured for the different pipetting systems used

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Fast, Precise, and Sterile Liquid Transfer in Cell Culture with Multipette® M4 /Repeater® M4

Impact of the pipetting system on cell counting 3 1.0 To evaluate the impact of the three pipetting systems on cell Eppendorf Xplorer

counting a manual air-cushion pipette (Eppendorf Research plus) with ep Dualfilter T.I.P.S., a positive displacement system 0.8 highest value (Multipette M4/Repeater M4) with Combitips advanced, and lowest value an electronic pipette controller (Easypet 3) with serological 0.6 pipettes were compared. The number of HEK 293 cells was determined using the CASY technology. 0.4 (OD 450 – 690 nm) Pipetting system Mean cell number CV Mean viability N = 30 0.2 Signal generated by viable cells Air-cushion 1.65 x 106 cells/mL 3.0 % 96.4 %

Positive displacement 1.62 x 106 cells/mL 2.5 % 96.0 % 0.0 0 150,000 300,000 450,000 600,000

Pipette controller 1.50 x 106 cells/mL 6.8 % 96.2 % Number of cells/well dispensed

Table 1: Cell counting results obtained with three different pipetting systems used to dis- pense 100 µL of cell solution (n=30) 4 1.0 As shown in Table 1, the level of cell viability is around 96 % Multipette M4/Repeater M4

for all conditions assessed. The cell count variability of the positive displacement dispenser and the air-cushion pipette is 0.8 highest value almost equivalent while the reproducibility obtained with the lowest value pipette controller is significantly lower. 0.6 Impact of the pipetting system on cell seeding 0.4 Based on the cell counting data, six cell suspensions of 1.5 x (OD 450 – 690 nm) 106 cells/mL were seeded into 24-well plates. The electronic 0.2 Xplorer pipette with ep Dualfilter T.I.P.S., the Multipette M4/ Signal generated by viable cells Repeater M4 with Combitips advanced, and the Easypet 3 with serological pipettes were used for cell transfer. 40 µL/ 0.0 well of WST-1 were added. WST-1 is cleaved to formazan by 0 150,000 300,000 450,000 600,000 cellular enzymes largely dependent on the NAD(P)H produc- Number of cells/well dispensed tion in viable cells and therefore directly correlates to their number (Fig. 3–5). 5 1.0 Conclusion Easypet 3

The results show that using a positive displacement system, 0.8 highest value such as the Multipette M4/Repeater M4 in combination with lowest value Combitips advanced, for cell culture applications, ensures reproducibility of the final results. Data obtained with the 0.6 dispenser are less variable than those obtained with air-cushion pipettes. Reproducibility of the final results will be affected 0.4 when a less precise instrument such as a pipette controller (OD 450 – 690 nm) is used. 0.2 Signal generated by viable cells Combining accuracy, precision, and contamination prevention, the Multipette M4/Repeater M4 in combination with Combitips 0.0 advanced represents a perfect alternative for all routine tasks 0 150,000 300,000 450,000 600,000 requiring a liquid handling device in cell biology labs. Number of cells/well dispensed

Literature Fig. 3 – 5: Signal curves generated by increasing numbers of cells. Three different pipetting systems were used for cell seeding: Xplorer (Fig. 3), Multipette M4/Repeater M4 (Fig. 4), [1] EN ISO 8655:2002, Parts 1– 6. Piston-operated volumetric apparatus. and Easypet 3 (Fig. 5). Beuth-Verlag, Berlin, Germany.

Readers’ service Multipette® M4 (US/CAN: Repeater® M4) • Ref. No. 268

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Solving the Aggregation Problem of HEK293 Cells ™ Using the New Brunswick S41i CO2 Incubator Shaker

STACEY WILLARD AND MA SHA, EPPENDORF, INC., ENFIELD, CT, USA

Abstract mine, 10 % FBS). For the 293/hTLR4-HA Western blot and immunofluorescence cells, the medium was supplemented analysis was used to confirm hTLR4 Human embryonic kidney 293 (HEK293) with blasticidin to maintain plasmid expression post adaptation using a cells are an excellent host for protein stability. Attachment cultures were mouse antibody raised against the HA expression, but the issue of cell clump- grown in T-75 flasks at 37 °C, 5 % CO tag (Data not shown). ing in large-scale suspension culture 2 on the static shelf of the New Brunswick has limited its use in bioprocess. Here, Results and discussion S41i (Figure 1). we show a successful example of the As Table 1 indicates, varying levels of adaptation of a human recombinant success were documented in each cat- protein-expressing HEK293 cell line egory using the tested media formula- from serum-supplemented attachment tions; a successful adaptation was only culture to serum-free single cell suspen- achieved if the cells retained high via- sion culture using the New Brunswick bility and grew to high densities in the S41i CO incubator shaker. By supporting 2 presence of blasticidin with no aggrega- the cultivation of adherent and suspen- tion under serum-free conditions. If cell sion cells simultaneously in the same clumping was severe (+++), or viability chamber and by allowing the evaluation was low, the culture was discontinued of multiple adaptation methods in par- and adjustments to the method were allel, the New Brunswick S41i is well made accordingly. For example, DMEM suited to evaluate adaption methods. with 1 % HI-FBS resulted in large cell Fig. 1: The New Brunswick S41i CO2 incubator shaker Introduction clumps of over 50 cells in suspension Suspension cell culture (Fig. 2A). Within the biopharmaceuticals market, HEK293 cells are among the most Adaptation to suspension culture was Therefore, after 48 h of culture, the versatile hosts for recombinant protein carried out using multiple methods formulation was adjusted to contain expression. These cells are capable of (Table 1, see next page) according to the anti-clumping agents such as Pluronic expressing large membrane proteins manufacturer’s protocol. Blasticidin F-68 and Anti clump A, and a new cul- that are often not properly expressed by was added to each media formulation ture was established from attachment even the most popular biopharmaceuti- at an initial dose of 5 μg/mL. cells. As outlined in Table 1, DMEM was cal production hosts such as Chinese not able to support suspension cell Cells were cultivated in 125 mL Erlen- hamster ovary (CHO) cells. Although an growth without aggregation in this ex- meyer flasks on the S41i shaking plat- excellent host for protein expression, periment. Another formulation, Pro293™ form at 125 rpm, 8 % CO , and 37 °C. the issue of cell clumping in large-scale 2 s-CDM™, was able to sustain the growth Cell density, % viability, and cell suspension culture of HEK293 cells has of 293/hTLR4-HA with serum supple- clumping were analyzed every 72 h. limited its use in bioprocess. Recently, mentation, however, when serum wean- many commercial entities have devel- Cell clumping was assigned a score ing was complete, the cells did not sur- oped specialty media formulations and from (−) indicating no clumping to vive for multiple passages (Fig. 3A). anti-clumping reagents to help combat (+++) representing large cell masses, The most successful adaptation meth- this issue. Here, we use a media array based on the relative size of the cell od using this strategy was Method 4 technique to evaluate the effectiveness clumps (Fig. 2). (EX-CELL® 293) which, after blasticidin of various anti-clumping methods using a commercially available human membrane protein-expressing HEK293 cell A B line. Materials and methods Details are described in the Application Note No. 339 at www.eppendorf.com/ applications. Attachment cell culture

HEK293 cells expressing hemaggluti- 400 µm 400 µm nin-tagged human Toll-like receptor 4 (hTLR4-HA) and untransfected HEK293 Fig. 2: The evaluation of clumping in suspension 293/hTLR4-HA cells: cells (control) were initially cultivated in A: Cells formed large aggregates containing > 50 cells, this culture received an aggregation score of (+++). B: Successful adaptation in which the culture showed no evidence of cell clumps greater than 2‑4 cells; this culture was standard media (DMEM, 4 mM L-gluta- given a (−) for aggregation.

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Solving the Aggregation Problem of HEK293 Cells ™ Using the New Brunswick S41i CO2 Incubator Shaker

Method Base Medium Formulation Aggregation? Viable with Blasticidin? Serum-free?

1 DMEM 0 % HI-FBS N/A No No

1 % HI-FBS +++ No No

1 % HI-FBS, Pluronic F-68 +++ No No

1 % HI-FBS, Anti-clumping agent A ++ No No

2 CD 293 As recommended +++ N/A Yes

3 293 SFM II No supplement − N/A Yes

5 µg/mL blasticidin − No Yes

1 % BSA, 5 µg/mL blasticidin − No Yes

4 EX-CELL® 293 No supplement − N/A Yes

10 µg/mL blasticidin − No Yes

5 µg/mL blasticidin − Yes Yes

5 Pro293™ s-CDM™ 5 % HI-FBS, 5 µg/mL blasticidin − Yes No

2.5 % HI-FBS, 5 µg/mL blasticidin − Yes No

1 % HI-FBS, 5 µg/mL blasticidin − Yes No

5 µg/mL blasticidin − No Yes

6 PeproGro HEK293 As recommended +++ N/A Yes

Table 1: Results of suspension cell culture with the formulations tested

A 293 II SFM B EX-CELL 293 4.5 100 4.0 100

90 90 4.0 3.5

3.5 80 80 3.0 70 70 cells/mL) cells/mL)

6 3.0 6 2.5 60 60 2.5 50 2.0 50 2.0 40 40 Viability (%) 1.5 Viability (%) 1.5 30 30 1.0 1.0 20 20

Viable cell density (x 10 Viable cell density (x 10 0.5 0.5 10 10

0.0 0 0.0 0 0 5 10 15 20 25 30 35 40 0 10 20 30 40 50 60 Time (Days) Time (Days) Viable cell density Viability Viable cell density Viability

Fig. 3: 293/hTLR4-HA cell adaptation to suspension culture. A: Cells were able to adapt to suspension culture without serum, but died upon addition of blasticidin (yellow line). B: Successful adaptation to single cell serum-free suspension culture. The yellow line denotes the addition of blasticidin. Western blot and immunofluorescence analysis showed that protein expression of suspension-adapted cells was not impacted by the adaptation process (data not shown). addition, resulted in virtually no cell known large-scale suspension culture methods in the New Brunswick S41i clumps and reproducible high cell den- aggregation issue in bioreactor condi- CO2 incubator shaker. This method cuts sities and viabilities without the pres- tions. down on upstream process development ence of serum (Fig. 3B). time since it can support adherent and In this work, we eliminated the clump- suspension cells simultaneously in the This method was deemed successful ing problem in our HEK293 cell line same chamber. for the adaptation of this cell line to se- prior to the bioreactor production stage, rum-free suspension culture, the other leveraging commercially available se- methods were halted at this stage. rum-free adaptation methods. We have shown that the adjustment of a mem- Conclusion brane protein-expressing HEK293 cell The number of FDA-approved biophar- line to clump-free serum-free suspension maceuticals produced in HEK293 cells culture can be accomplished by simul- Readers’ service ™ New Brunswick S41i CO2 Incubator Shaker • Ref. No. 253 has been low, partially due to the well- taneously testing multiple adaptation

Your local distributor: www.eppendorf.com/contact Eppendorf AG · 22331 Hamburg · Germany · e-mail: [email protected] · www.eppendorf.com LIQUID HANDLING EXCELLENCE REWARDED · NEWS 9

CHRISTIANE MARKAU, EPPENDORF AG

Liquid Handling Close-up Excellence Rewarded Fill 96 Wells at once with epMotion® 96

The Eppendorf epMotion 96 is a semi- automated electronic pipette for fast and In 1961, Eppendorf launched the first “Eppendorf partnered with experts in precise parallel 96-channel microplate piston-stroke pipette and the microtube physiotherapy and ergonomics to introduce processing in a volume range from 0.5 to to laboratory work, and since then liquid several new product features to reduce the 300 μL. An epMotion 96 will help you save handling has been unthinkable without risk of repetitive strain injury.” (More info time and reduce your RSI (Repetitive Strain Eppendorf products. For more than five at www.eppendorf.com/physiocare.) Injury) risk. Running an epMotion 96 is decades, we have been committed to “…, Eppendorf sets industry standards in convenient and intuitive! innovation and developing high-quality, liquid handling and offers comprehensive reliable products. The typical “Eppendorf Convenient system control systems that make workflows in laborato- drive” to relentlessly improve our prod- ries more efficient and noticeably ease the >> Control your epMotion 96 with an ucts with novel smart features has re- burden of routine tasks.” (More info at Apple® iPod with high quality touch sulted in multiple pipette and dispenser www.eppendorf.com/epmotion.) screen lines and a broad portfolio of automated >>epMotion 96 also accessible by other workstations for ultimate flexibility and iOS® 7 devices (iPhone®, iPad®) via WiFi customization to our customers’ needs.

For its commitment to innovation and delivering customer-focused products to meet diverse end-user needs, Eppendorf About Frost & Sullivan received the “2015 North American New Product Innovation Award for Liquid Frost & Sullivan (www.frost.com) indus- Handling” from Frost & Sullivan at the try analysts compare market participants “Excellence in Best Practices Awards and identify best-practice companies by Gala” in San Diego, CA, USA, in March measuring performance through in-depth 2015. interviews, analysis, and secondary research. The companies demonstrating The laudators: outstanding achievement and superior “One of the main differentiators of performance in areas such as leadership, Eppendorf’s pipettes is its focus on technological innovation, customer ser- ergonomic features. Recognizing the vice, and strategic product development need to reduce hand strain caused by are recognized with prestigious Best manual pipetting for long periods of Practices Awards in a variety of regional time” … and global markets. Intuitive operation

>> Software based on Eppendorf Xplorer® electronic pipettes; provided as free Apple app >>Intelligent preset applications >> Individual parameters for aspiration or dispensing speed depending on your need >> Simple programming of multi-step 2015 pipetting tasks For more features and applications see www.eppendorf.com/epmotion or the brochure. NORTH AMERICAN LIQUID HANDLING NEW PRODUCT INNOVATION AWARD epMotion® 96 • Ref. no. 275 North American Liquid Handling New Product Innovation Award eppendorf.com/frost 10 STRAIGHT FROM THE LAB · WORKFLOW SOLUTIONS FOR MICROBIOLOGY AND FERMENTATION

TANJA MUSIOL AND CHRISTIANE SCHLOTTBOM, EPPENDORF AG Workflow Solutions for Microbiology and Fermentation

Microorganisms such as bacteria, yeasts, and fungi are employed in numerous applications in the pharmaceutical, biochemical, and food industries. Efficient methods for their cultivation, modification, and analysis are crucial for the production of foods, drugs and vaccines, enzymes, and biofuels as well as biopolymers. Eppendorf pre- mium products have guided these steps and other processes for more than 70 years. Our innovative technologies facilitate daily work for our users and provide optimum conditions for reproducible and scalable results.

Microbiology (Prokaryotic cells) Electroporator Centrifuges Tubes Thermo mixing devices

Transformation

Shakers Bioreactors Pipettes and tips Deepwell plates Cultivation

Preparation and purification Centrifuges Pipettes and tips Thermo mixing devices Automation

Detection and amplification

Photometers Cuvettes Thermocyclers PCR consumables

Storage

Eppendorf offers a wide variety of laboratory instruments and accessories for Deepwell plates Sealing options Concentrator Freezers applications in the field of microbiology.

Transformation Cultivation Preparation and analysis Preparation of DNA allows targeted ma- Following transformation, the microorgan- The plasmid DNA is isolated, purified, nipulation of microorganisms and cells. isms are cultured and propagated in spe- and analyzed. Purification is frequently For example, electroporation using the cialized media. For this step, Eppendorf performed using respective reagent kits. Eppendorf Eporator® can render the cell offers the New Brunswick™ Innova® shak- The DNA is isolated from the cells and membrane of a microorganism transient- ers which feature flexible options for purified via chromatography columns ly permeable by applying an electrical shaking as well as temperature control. and the addition of buffers containing impulse. The resulting permeability al- salts and other reagents. These options are programmable, serv- lows the introduction of a vector, which ing the individual requirements of the The reagents are drawn through the col- carries the functions of choice, into the application. The flexible layout of the umn material by either centrifugation or microorganism. inner compartment allows cultivation in the application of a vacuum. In a final The very compact Eporator enables fast different vessels and volumes. Following step, the purified DNA is harvested from and effective transfer of DNA under re- incubation, these cultures often serve as the column using either one of these two producible conditions in an intuitive and the basis for subsequent plasmid prepa- methods. If centrifugation is the method user-friendly manner. rations. of choice, it is possible that the lids of WORKFLOW SOLUTIONS FOR MICROBIOLOGY AND FERMENTATION · STRAIGHT FROM THE LAB 11

the tubes in which the reagent or purified DNA are to be collected will tear off due to shear forces. This is not the case in the special Eppendorf Kit rotor® of the microcentrifuges 5424/R, 5427 R, and 5430/R: Its extended rim supports the tube lids and prevents them from shearing off. In cases where many samples are pro- cessed simultaneously, columns in 96-well plate format are often the pre- ferred choice. One of the methods used to draw reagents through the columns includes a vacuum chamber. Since a number of different reagents are involved in the process, an electronic pipette such as the Eppendorf Xplorer® or Xplorer plus, or a 96-well pipetting system such as the Eppendorf epMotion® 96, can sim- plify this workflow significantly. In fact, the larger Liquid Handling Workstation DASbox Mini Bioreactor System for microbial applications epMotion 5075v is capable of automating the entire process. plate. In order to achieve these optimal working volume, thus covering the wide conditions, excellent block homogeneity spectrum from process development all must be complemented with effective the way to production. evaporation protection during the PCR The DASbox® Mini-Bioreactor System run. To this end, Eppendorf’s portfolio and the DASGIP® Parallel Bioreactor includes two new HeatSealers, the handy Systems accelerate strain and media HeatSealer S100 and the HeatSealer S200 optimization, as well as process devel with flexible, programmable sealing opment, by parallel process control. parameters. Intelligent software solutions deliver comprehensive options for data and information management, Design of Experiments (DoE), and integration of external analyzers (see Application Notes pages 3 – 4). Thanks to the BioBLU® f Single-Use Vessels, users in the field of microbiology now benefit from the advantages of single-use technology. These single-use bioreactors, specifically geared towards the needs of microbial cultivation, can be operated with the Intuitive handling concept for fast and easy operation: elec- Eppendorf DASbox, DASGIP, and BioFlo® tronic pipettes Eppendorf Xplorer and Xplorer plus benchtop controllers (see also pages Detection and amplification 6 – 7). Adaptor kits for instruments by third party suppliers are also available. Once the DNA is isolated and purified, analysis is next. PCR analysis provides Flexible BioFlo sterilize-in-place (SIP) first results about success or failure of fermentors allow scale-up to pilot and Modern networking: Your Mastercycler nexus informs you via the manipulation. In addition to fast e-mail once your PCR is completed. production scale. They feature a com- reaction times, the Mastercycler® nexus pact design and numerous upgradable From small to big – microorganisms in gradient, with its gradient function, options which allow adaptation to the industrial production offers the option of determining the demands of the process. annealing temperatures of primer pairs Genetically modified microbial strains More information in a single experiment. Once the PCR is are frequently employed in industrial established, experimental conditions production. Eppendorf offers scalable www.eppendorf.com/microbiology should be reproducible in each well of the fermentors from 60 mL up to 2,400 L 12 STRAIGHT FROM THE LAB · AUTOMATED NGS SAMPLE PREPARATION

CARSTEN BUHLMANN, EPPENDORF AG

Automated NGS Tip Sample Preparation Eppendorf App The Eppendorf App provides you with product information and a variety of useful tools for your daily routine in the lab – optimized for mobile devices. Illumina® TruSeq® Stranded mRNA Library Prep Kit New products >> Stay informed about news in the The Short Protocol No. 02 describes Eppendorf portfolio. the configuration and preprogrammed methods for the automated construction Product catalog of 8, 16, or 24 sequencing ready libraries >> Browse our complete digital catalog with from 100 – 1,000 ng total RNA with the lab and bioprocess products. TruSeq Stranded mRNA kit. The overall >> Download the catalog and use it even hands on time is less than one hour, the without an active internet connection. total run time of the entire procedure is Tools ~11.5 hours for 24 samples. >> Find a wide range of helpful lab tools, Illumina® TruSeq® Nano DNA Library like timer or Standard Genetic Code. Prep Kit Calculators With a built-in Eppendorf ThermoMixer® The Short Protocol 05 describes the con- >> User-friendly calculators for many the epMotion® 5075t and 5075 TMX (pre- figuration and preprogrammed methods standard applications. decessor model) are the most flexible for the automated construction of 8, 16, News members of the epMotion family of auto- or 24 sequencing ready libraries from >> All news about Eppendorf and information mated pipetting systems. Customized 100 or 200 ng DNA (depending on the on international trade fair participation. “Illumina Qualified” methods for selected insert size) with the TruSeq Nano DNA kit. Illumina®* NGS Library Preparation Kits Contact & Feedback The overall hands on time is less than 1.5 have been developed for use in your >> Contact Eppendorf, order product hours, the total run time on the epMotion epMotion 5075t/TMX. samples, or request a visit from an 5075 TMX for 24 samples is ~10 hours. Eppendorf expert. The respective Short Protocols, method After Covaris® fragmentation of the sam- >> Help us optimize our products and and labware files can be downloaded at ple DNA, the entire library construction services even further. www.eppendorf.com/epmotionvip. is automated on the epMotion. Download now at For the MiSeq® and HiSeq® NGS systems, How to run your Illumina® NGS method www.eppendorf.com/eppendorf-app Illumina offers a variety of sample prep- on your epMotion® aration kits used to convert DNA or RNA Or use the QR code! It’s easy: Just download and transfer the samples into sequencing ready libraries, method and labware files to your epMotion a procedure that includes many steps and – and start. Depending on sample type, is costly. Due to the complexity of the amount, and pooling strategy, optimiza- library construction methods, automation tion of the methods might be necessary. is regarded as highly advantageous. Further NGS sample preparation methods Illumina® TruSeq® Stranded Total RNA are available on demand or in preparation. Library Prep Kit The Short Protocol No. 01 describes the configuration and preprogrammed

methods for the automated construction *Illumina (www.illumina.com) is a leading developer, manu- of 8, 16, or 24 sequencing ready libraries facturer, and marketer of life science tools and integrated systems for large-scale analysis of genetic variation and from 100-1,000 ng total RNA with the function. The methods described are intended for molecular TruSeq Stranded Total RNA kit. The over- research applications. They are not intended, verified or validated, for use in the diagnosis of disease or other human all hands on time is less than one hour, health conditions. the total run time of the entire procedure ® is ~11.5 hours for 24 samples. epMotion Family Brochure • Ref. no. 254 RESEARCH PRIZES: WINNERS VISIT EPPENDORF · NEWS 13

BERRIT HOFF AND CAROLYN TAUBERT, EPPENDORF AG Research Prizes: Winners Visit Eppendorf

Dr. Thomas Wollert, Research Group saved. I need a financial buffer since my appointment at the Leader Molecular Membrane and Max Planck Institute is only temporary. Organelle Biology at the Max Planck More information www.eppendorf.com/award Institute of Biochemistry in Martins ried, Germany, won the 20,000 € Eiman Azim, Ph.D., Postdoctoral Re- Eppendorf Award for Young European search Fellow at Columbia University, Investigators 2015 for his ground- New York, and winner of the 2014 breaking work in reconstituting com- Eppendorf & Science Prize for Neuro- plex intracellular membrane events biology (see BioNews 42), also visited in the test tube using artificial mem- Eppendorf in the summer of 2015. branes and purified components. His Eiman Azim’s work has provided fun- experiments have paved the way for damental new insights into the neural understanding key steps in autophagy, mechanisms that enable skilled limb a fundamental process required for movements to be both smooth and the clearance of damaged cell parts precise. He took some time to answer Thomas Wollert giving a lecture at Eppendorf headquarters in all eukaryotic cells. our questions. BioNews: How did you feel when you heard that you had won? BioNews: Can you tell us what attracted Eiman Azim you to the prize? Thomas Wollert: I was absolutely surprised and happy at the same time! This award honors the hard work of my laboratory Eiman Azim: The prize attracted me because the application and represents a major acknowledgement of our efforts in essay provided me with a unique opportunity to step back and aiming to decipher how cells maintain their homeostasis by explore how my work might relate to a more general audience. autophagy. I am particularly grateful that our unusual bottom- It encouraged me to take a creative approach to describing my up approach, which is based on biophysics in combination science, and it was aimed at the intersection of neuroscience with biochemistry and cell biology, is being recognized by this and molecular biology, which is exactly where I spend my time. award. BN: Do you think the Eppendorf & Science Prize will help your BN: In summer 2015 you visited Eppendorf headquarters and career? also Eppendorf’s pipette and consumables production plants. EA: Absolutely. Applying for this prize made me think deeply How was your trip to Northern Germany? about what it is about motor control that interests me most TW: It was interesting to have insight into a company the and might be most relevant to advancing our understanding of products of which I have been familiar with since my very first the brain. And winning this prize certainly gave my work a lot work experience in a lab. I was especially excited to learn how of exposure, which has been a huge help throughout this past pipettes and the famous “Eppis” are manufactured. It was also year as I have been navigating the difficult path of finding an great to see the pipette calibration and quality control. The trip independent position and securing funding. I’m extremely to the consumables production plant was another highlight. grateful to Eppendorf and the journal Science for this honor. Injection molding is probably something that everybody has More information www.eppendorf.com/prize heard of before – but seeing this process in reality was both exciting and informative. I was really impressed by how pipette tips and other consumables are made – in incredible quantities and with such a high degree of automation. BN: How did you spend your prize money? TW: First of all I took my lab team on a paddle tour on the Isar River. Ten of us paddled from Bad Toelz to Wolfratshausen near Munich where we had some great food and beer in a beer garden. The rest of the prize money will be put aside and 14 TIP · FOR ADDITIONAL INFORMATION, PLEASE CONSULT THE OPERATING MANUAL!

BERRIT HOFF, EPPENDORF AG For Additional Information, Please Consult the Operating Manual!

Cross your heart – when was the last time you sat down with a cup of coffee or tea to read an operating manual from cover to cover? Never? In this case, you are not alone. Most users only reach for the manual to find out how to set up an instrument or find a solution to a problem. But an Eppendorf manual has so much more to offer, explains Frank Thormählen (Executive Director Technical Writing at Eppendorf).

Mr. Thormählen, what are the hallmarks of How does your staff approach a new account those laws and standards – a good operating manual? project? country-specific to a certain extent – which apply to the instruments and Frank Thormählen: It provides the user FT: We collaborate closely with the dif- consumables described. All standards with a solution to their problem, as ferent departments whose requirements are listed in the CE declaration of the simply and quickly as possible. This is are incorporated in a basic manuscript. instrument. Since the manual bears supported by a good chapter structure, This “rough diamond” is then subjected to legal significance, we have to meet high a clear index, completeness, action- our finishing touches. This includes care- demands with respect to documentation oriented titles, as well as professional ful terminology work, generation of the of changes as well as archiving of ver- illustrations. A sophisticated task for all necessary illustrations by our graphic sions. colleagues involved! artists, “hands on” time with the product in order to confirm that everything is de- Tip: For downloading any Eppendorf Do Eppendorf clients read your scribed completely and correctly, and operating manual, please visit instructions? last but not least, the safety instructions. www.eppendorf.com/manuals. FT: As a rule, Eppendorf products are Which technologies are available to you? very intuitive, so that users do not have to consult the manual for every basic FT: We use modern tools supported by operation. But it has so much more to databases. In order to remain linguisti- offer! For example, besides information cally correct and consistent, as well as on cleaning and handling of accessories, adhere to certain linguistic rules, also in a centrifuge manual specifies which ro- regard to readability, we employ the Trademarks Information Amazon® is a registered trademark of Amazon Tech, Inc., USA. tors and adapters can be used for which support of an editing system and a ter- Apple®, iPod®, iPad®, and iPhone® are registered trademarks of Apple, Inc., USA. CASY® and Roche® are registered trade- containers. It further contains details minology tool. marks of Roche Diagnostics Co., USA. Covaris® is a registered about radii and g-forces, which play a trademark of Covaris, Inc., USA. EX-CELL® is a registered We then have the completed work trans- trademark of Sigma-Aldrich Co. LLC., USA. Flownamics® and role in the correct implementation of an Seg-Flow® are registered trademarks of Flownamics Ana- lated into up to 26 languages! Our internal lytical Instruments, Inc., USA. HiSeq®, Illumina®, MiSeq®, and application, as well as for documentation TruSeq® are registered trademarks of Illumina, Inc., USA. iOS® Translation Management takes care of is a trademark or registered trademark of Cisco in the U.S. and of client-specific processes. The number other countries and is used by Apple under license. MEBAK® that. Of course we do not translate every- is a registered trademark of Mitteleuropäische Brautech- of downloaded documents from the nische Analysenkommision e.V., Germany. Mettler-Toledo® thing ourselves, but we use the same is a registered trademark of Mettler-Toledo AG, Switzerland. Eppendorf website speaks for the frequent FlowWeb™ and FlowFraction™ are trademarks of Flownamics translation tool as our external translators. Analytical Instruments, Inc., USA. s-CDM™ and Pro293™ are use of our manuals! trademarks of Lonza AG Corporation, Switzerland.

The final step consists of data entry into ® ® ® What are the qualifications of your staff? Eppendorf , the Eppendorf logo, BioBLU , Biopur , Combi a product information database which tips advanced®, Easypet®, ep Dualfilter T.I.P.S.®, epMotion®, Eppendorf BioSpectrometer®, Eppendorf Eporator®, Eppen- FT: We have a broad spectrum of expertise feeds our websites, the Eppendorf App dorf Kit rotor®, Eppendorf Reference®, Eppendorf Thermo- Mixer®, Eppendorf Tubes®, Eppendorf Xplorer®, epPoints®, and experience, ranging from university- (also see page 12), and more. the epServices® logo, epT.I.P.S.®, Mastercycler®, Multipette®, and Repeater® are registered trademarks of Eppendorf AG, educated technical editors to linguists Germany. Eppendorf Quality™ and New Brunswick™ are trade- Which legal requirements exist? marks of Eppendorf AG , Germany. DASbox®, DASGIP®, and to engineers. We continue to educate DASware® are registered trademarks of DASGIP Informa- ourselves in order to stay abreast of the FT: Standard EN 82079 governs operat- tion and Process Technology GmbH, Germany. BioFlo® and Innova® are registered trademarks of Eppendorf, Inc., USA. latest developments. ing manuals. Our manuals also take into U.S. Design Patents are listed on www.eppendorf.com/ip. PRIZE COMPETITION · SERVICE 15

Your Chance to Win!

The solution of the prize competition of BioNews No. 42 was You can either send us an e-mail to [email protected], “Cell Biology Workflow”. Ewa Sztupecka (Kazimierz Wielki or participate online at www.eppendorf.com/bn-service. University, Bydgoszcz, Poland) won the first prize. All correct answers will be considered for a prize. Winners will Good luck in our new competition! be notified in writing. Cash payment of the prize is not possible. No recourse to legal action. The judges’ decision is final. How to find out the solution: Simply arrange all letters in the Eppendorf employees and their families may not participate. light gray boxes of the crossword in the correct order. Send us The winner of the first prize will be published in BioNews No. 46. the solution until June 30, 2016.

1 2 3 4 5 6 7 8 9 st 10 11 12 13 14 15 1 Prize: ® 16 17 18 19 1 Eppendorf Reference 2

20 21 22 23 Pipette (single-channel, adjustable-volume) of your 24 25 26 27 choice 28 29 30 31 32 nd th 33 34 35 36 37 2 to 5 Prize: 38 39 1 Amazon® Voucher

40 41 42 43 44 45 worth 50.00 EUR

46 47 48 49 6th to 15th Prize: 50 51 52 400 bonus epPoints® each 53 54 55 56 57 58 (epPoints registration required) 59 60

ACROSS DOWN 2 Unsolicited e-mail 33 Chemical symbol for manganese 1 Short form of Robert 30 Requirement for manufacturers of e.g. 6 Spanish word for coast 34 100 liters (abbrev.) 3 Piece or segment of a whole pharmaceutical products (abbrev.) 10 Preposition 35 ISO country code for Ghana 4 Chemical symbol for silver 31 Non-governmental organization 12 Polysaccharide, used in 37 Advertisement (abbrev.) 5 Male first name (abbrev.) electrophoresis 38 ISO country code for Austria 6 Police officer (informal) 36 Technique for separating/measuring 14 The capital is Lima (ISO country 39 ISO country code for Guadeloupe 7 Chemical symbol for osmium components in a chemical mixture code) 40 Can be consulted for additional 8 Photograph in brownish tone (abbrev.) 16 Alcoholic beverage information 9 Program run on mobile devices 39 Good Laboratory Practice (abbrev.) 18 Requiescat in pace (abbrev.) 44 Ill-fated king, father of three daughters 11 Biochemical process 41 Healthcare professional 19 Eppendorf Tubes® are made of this 46 Plant of the lily family 13 2nd largest city of Brazil 42 ISO country code for Anguilla thermoplastic polymer (abbrev.) 47 Open Platform Communications 15 Publisher of BioNews 43 Sum, entirety 20 American painter (1903 –1970) (abbrev.) 17 Goddess of the dawn 45 Chemical symbol for argon 22 Helpful hint 49 Rhesus factor (abbrev.) 21 AC/DC found the highway to this 48 Sheet of paper in a book 24 Name prefix of ships of the Royal 50 Feathered vertebrate place 52 Hot beverage Navy 51 Chemical symbol for tantalum 23 Graphics file format (abbrev.) 55 Chemical symbol for rhenium 26 First name of Henry VIII's second 53 To suffer loss 24 Turkish steambath 56 Chemical symbol for magnesium wife 54 Piece broken off or detached 25 US State in the Pacific Northwest 57 No name (abbrev.) 28 ISO country code for the United 59 Roman emperor (abbrev.) 58 ISO country code for Trinidad and Arab Emirates 60 Tasteful in style and design 27 Dance cardio fitness technique Tobago 29 Motto, catch phrase 29 Part of the body 32 Spirit flavored with Juniperus

Solution hint for prize competition of BioNews No. 44:

U I F

Send us the solution until June 30, 2016, via e-mail to [email protected], or participate online at www.eppendorf.com/bn-service. want new ABN 4413020 technologies?

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