How Pipette Tips Influence Assay Results
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No. 44 – 2016 How Pipette Tips Influence Assay Results (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 recom- mended 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 fer- mentation are introduced on pages 10 –11. Starting with this issue, we will discontinue the traditional service fax. You can down- load 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.