Comparison of Count Reproducibility, Accuracy, and Time to Results
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Introduction to Flow Cytometry Principles Data Analysis Protocols Troubleshooting
Flow Cytometry ipl.qxd 11/12/06 11:14 Page i Introduction to Flow Cytometry Principles Data analysis Protocols Troubleshooting By Misha Rahman, Ph.D. Technical advisors Andy Lane, Ph.D. Angie Swindell, M.Sc. Sarah Bartram, B.Sc. Your first choice for antibodies! Flow Cytometry ipl.qxd 11/12/06 11:14 Page ii Flow Cytometry ipl.qxd 11/12/06 11:14 Page iii Introduction to Flow Cytometry Principles Data analysis Protocols Troubleshooting By Misha Rahman, Ph.D. Technical advisors Andy Lane, Ph.D. Angie Swindell, M.Sc. Sarah Bartram, B.Sc. Flow Cytometry ipl.qxd 11/12/06 11:14 Page 2 Preface How can I explain what flow cytometry is to someone that knows nothing about it? Well, imagine it to be a lot like visiting a supermarket. You choose the goods you want and take them to the cashier. Usually you have to pile them onto a conveyor. The clerk picks up one item at a time and interrogates it with a laser to read the barcode. Once identified and if sense prevails, similar goods are collected together e.g. fruit and vegetables go into one shopping bag and household goods into another. Now picture in your mind the whole process automated; replace shopping with biological cells; and substitute the barcode with cellular markers – welcome to the world of flow cytometry and cell sorting! We aim to give you a basic overview of all the important facets of flow cytometry without delving too deeply into the complex mathematics and physics behind it all. For that there are other books (some recommended at the back). -
Cell Counting and Viability Assessment of 2D and 3D Cell
Piccinini et al. Biological Procedures Online (2017) 19:8 DOI 10.1186/s12575-017-0056-3 METHODOLOGY Open Access Cell Counting and Viability Assessment of 2D and 3D Cell Cultures: Expected Reliability of the Trypan Blue Assay Filippo Piccinini1*† , Anna Tesei1†, Chiara Arienti1 and Alessandro Bevilacqua2,3 Abstract Background: Whatever the target of an experiment in cell biology, cell counting and viability assessment are always computed. The Trypan Blue (TB) assay was proposed about a century ago and is still the most widely used method to perform cell viability analysis. Furthermore, the combined use of TB with a haemocytometer is also considered the standard approach to estimate cell population density. There are numerous research articles reporting the use of TB assays to compute cell number and viability of 2D and 3D cultures. However, the literature still lacks studies regarding the reliability of the TB assay in terms of assessment of its repeatability and reproducibility. Methods: We compared the TB assay's measurements obtained by two biologists who analysed 105 different samples in double-blind for a total of 210 counts performed. We measured: (a) the repeatability of the count performed by the same operator; (b) the reproducibility of counts performed by the two operators. Results: There were no significant differences in the results obtained with 2D and 3D cell cultures: we estimated an approximate variability of 5% when the TB assay was used to assess the viability of the culture, and a variability of around 20% when it was used to determine the cell population density. Conclusions: The main aim of this study was to make researchers aware of potential measurement errors when TB is used with a haemocytometer for counting and viability measurements in 2D and 3D cultures. -
Antimicrobial Effect of Zophobas Morio Hemolymph Against Bovine
microorganisms Article Antimicrobial Effect of Zophobas morio Hemolymph against Bovine Mastitis Pathogens Mengze Du y, Xiaodan Liu y, Jiajia Xu , Shuxian Li, Shenghua Wang, Yaohong Zhu and Jiufeng Wang * Department of Veterinary Clinical Sciences, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China; [email protected] (M.D.); [email protected] (X.L.); [email protected] (J.X.); [email protected] (S.L.); [email protected] (S.W.); [email protected] (Y.Z.) * Correspondence: [email protected]; Tel.: +86-1355-221-6698 These authors contributed equally to this work. y Received: 2 September 2020; Accepted: 25 September 2020; Published: 28 September 2020 Abstract: Coliforms and Staphylococcus spp. infections are the leading causes of bovine mastitis. Despite extensive research and development in antibiotics, they have remained inadequately effective in treating bovine mastitis induced by multiple pathogen infection. In the present study, we showed the protective effect of Zophobas morio (Z. morio) hemolymph on bovine mammary epithelial cells against bacterial infection. Z. morio hemolymph directly kills both Gram-positive and Gram-negative bacteria through membrane permeation and prevents the adhesion of E. coli or the clinically isolated S. simulans strain to bovine mammary epithelial (MAC-T) cells. In addition, Z. morio hemolymph downregulates the expression of nucleotide-binding oligomerization domain (NOD)-like receptor family member pyrin domain-containing protein 3 (NLRP3), caspase-1, and NLRP6, as well as inhibits the secretion of interleukin-1β (IL-1β) and IL-18, which attenuates E. coli or S. simulans-induced pyroptosis. Overall, our results suggest the potential role of Z. morio hemolymph as a novel therapeutic candidate for bovine mastitis. -
The Past, Present and Future of Flow Cytometry in Central Nervous System Malignancies
Review The Past, Present and Future of Flow Cytometry in Central Nervous System Malignancies Evrysthenis Vartholomatos 1, George Vartholomatos 2, George A. Alexiou 1,3 and Georgios S. Markopoulos 1,2,* 1 Faculty of Medicine, Neurosurgical Institute, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece; [email protected] (E.V.); [email protected] (G.A.A.) 2 Haematology Laboratory-Unit of Molecular Biology, University Hospital of Ioannina, 45110 Ioannina, Greece; [email protected] 3 Department of Neurosurgery, University of Ioannina, 45110 Ioannina, Greece * Correspondence: [email protected] Abstract: Central nervous system malignancies (CNSMs) are categorized among the most aggressive and deadly types of cancer. The low median survival in patients with CNSMs is partly explained by the objective difficulties of brain surgeries as well as by the acquired chemoresistance of CNSM cells. Flow Cytometry is an analytical technique with the ability to quantify cell phenotype and to categorize cell populations on the basis of their characteristics. In the current review, we summarize the Flow Cytometry methodologies that have been used to study different phenotypic aspects of CNSMs. These include DNA content analysis for the determination of malignancy status and phenotypic characterization, as well as the methodologies used during the development of novel therapeutic agents. We conclude with the historical and current utility of Flow Cytometry in the field, and we propose how we can exploit current and possible future methodologies in the battle against this dreadful type of malignancy. Citation: Vartholomatos, E.; Keywords: central nervous system malignancies; flow cytometry; glioblastoma; intraoperative flow Vartholomatos, G.; Alexiou, G.A.; cytometry; phenotypic analysis; DNA content analysis Markopoulos, G.S. -
Preliminary Experiment for Cell Count Using Flow Cytometry1 Yuki Morono,2 Jens Kallmeyer,2 Fumio Inagaki,2 and the Expedition 329 Scientists2
D’Hondt, S., Inagaki, F., Alvarez Zarikian, C.A., and the Expedition 329 Scientists Proceedings of the Integrated Ocean Drilling Program, Volume 329 Preliminary experiment for cell count using flow cytometry1 Yuki Morono,2 Jens Kallmeyer,2 Fumio Inagaki,2 and the Expedition 329 Scientists2 Chapter contents Abstract Abstract . 1 One of the major challenges in microbial ecology is to evaluate the accurate number of living cells in a natural environment. Cell Introduction . 1 count using flow cytometry is a powerful, high-throughput tech- Principles of fluorescent wavelength–based cell discrimination. 2 nique that has widely been used for aquatic habitats but not for sedimentary environments because mineral grains interfere with Standardization of flow cytometry cell count protocol for South Pacific Gyre sediment . 2 cell detection. During Integrated Ocean Drilling Program Expedi- Results and discussion. 3 tion 329, we tested several sample preparation methods for on- board cell counting using flow cytometry with various pelagic Conclusion and future prospects . 6 sediments from the South Pacific Gyre. The cell numbers acquired Acknowledgments. 6 from shallow sediments are almost consistent with microscopic References . 6 direct counts (i.e., ~105 cells/cm3). Yet, the method needs im- Figures . 7 provement and standardization to reduce the background signal and to lower the minimum detection limit for deep sedimentary habitats with very low cell densities (i.e., <1000 cells/cm3). Introduction Detection and enumeration of microbial life in marine subsurface environments provides primary information for deciphering the extent and habitability of Earth’s biosphere. Hence, accurate and precise knowledge of microbial cell abundance at high spatial res- olution is a major challenge for the exploration of subsurface life. -
A Method for High Throughput Determination
Hazan et al. BMC Microbiology 2012, 12:259 http://www.biomedcentral.com/1471-2180/12/259 METHODOLOGY ARTICLE Open Access A method for high throughput determination of viable bacteria cell counts in 96-well plates Ronen Hazan1,2,3,4†, Yok-Ai Que1,2,3†, Damien Maura1,2,3 and Laurence G Rahme1,2,3* Abstract Background: There are several methods for quantitating bacterial cells, each with advantages and disadvantages. The most common method is bacterial plating, which has the advantage of allowing live cell assessment through colony forming unit (CFU) counts but is not well suited for high throughput screening (HTS). On the other hand, spectrophotometry is adaptable to HTS applications but does not differentiate between dead and living bacteria and has low sensitivity. Results: Here, we report a bacterial cell counting method termed Start Growth Time (SGT) that allows rapid and serial quantification of the absolute or relative number of live cells in a bacterial culture in a high throughput manner. We combined the methodology of quantitative polymerase chain reaction (qPCR) calculations with a previously described qualitative method of bacterial growth determination to develop an improved quantitative method. We show that SGT detects only live bacteria and is sensitive enough to differentiate between 40 and 400 cells/mL. SGT is based on the re-growth time required by a growing cell culture to reach a threshold, and the notion that this time is proportional to the number of cells in the initial inoculum. We show several applications of SGT, including assessment of antibiotic effects on cell viability and determination of an antibiotic tolerant subpopulation fraction within a cell population. -
A Deep Profiler's Guide to Cytometry
TREIMM-937; No. of Pages 10 Review A deep profiler’s guide to cytometry 1 1 2 2 Sean C. Bendall , Garry P. Nolan , Mario Roederer and Pratip K. Chattopadhyay 1 Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA 2 ImmunoTechnology Section, Vaccine Research Center, NIAID, NIH, Bethesda, MD, USA In recent years, advances in technology have provided us and data analysis, and have led to state-of-the-art 20-pa- with tools to quantify the expression of multiple genes in rameter flow cytometers. Concomitant with this develop- individual cells. The ability to measure simultaneously ment, our understanding of immunology and stem cell multiple genes in the same cell is necessary to resolve biology has matured tremendously with the discovery of the great diversity of cell subsets, as well as to define scores of functionally diverse cell populations. Here, we their function in the host. Fluorescence-based flow cyto- review the development and highlight applications of poly- metry is the benchmark for this; with it, we can quantify chromatic flow cytometry (PFC, 6+ colors). In addition, we 18 proteins per cell, at >10 000 cells/s. Mass cytometry is review recent advances in a next-generation, ‘post-fluores- a new technology that promises to extend these capa- cence’ single-cell technology termed mass cytometry, which bilities significantly. Immunophenotyping by mass spec- is theoretically capable of measuring 70–100 parameters. trometry provides the ability to measure >36 proteins at Both fluorescence and mass cytometry have unique and a rate of 1000 cells/s. -
Flow Cytometric Approach to Probiotic Cell Counting and Analysis
APPLICATION OVERVIEW Flow Cytometric Approach to Probiotic Cell Counting and Analysis Data provided by Dharlene Tundo, Department of Quality Control, VitaQuest International IN THIS PAPER YOU WILL Compare and contrast See a detailed staining Learn how to set up the culture-based and flow protocol and gating strategy flow cytometer for optimal cytometry based workflows for enumerating live versus detection of bacterial strains. for probiotic cell enumeration dead probiotic cells. Introduction Probiotics were historically defined as substances secreted by one microorganism that promote the growth of another. The history of probiotics goes parallel with the evolution of the human race and can be traced back to the ancient times, nearly 10,000 years ago (1). Extensive research in recent years has exploded our understanding of the 100 trillion gut resident microbial cells contribute to health and disease (2). Indeed, industries devoted to leveraging beneficial organisms to restore balance to the gut microbiota have developed and continue to flourish. One manufacturer is VitaQuest International, one of the largest custom contract manufacturers of nutritional supplements in the United States. They produce a range of probiotic containing supplements manufactured at large scale. Over twenty different bacterial strains from within the Lactobacillus and Bifidobacterium species, are handled in the formulation of different products. The complexity of the manufacturing process along with increased demand, and strict criteria for quality, potency, and safety in accordance to FDA regulations necessitates that manufacturers continue to improve production processes that increase productivity and efficiency. A key method used to ensure product quality is the enumeration of probiotic organisms contained in the product. -
Simplified White Blood Cell Differential: an Inexpensive, Smartphone- and Paper-Based Blood Cell Count
Simplified White Blood Cell Differential: An Inexpensive, Smartphone- and Paper-Based Blood Cell Count Item Type Article Authors Bills, Matthew V.; Nguyen, Brandon T.; Yoon, Jeong-Yeol Citation M. V. Bills, B. T. Nguyen and J. Yoon, "Simplified White Blood Cell Differential: An Inexpensive, Smartphone- and Paper-Based Blood Cell Count," in IEEE Sensors Journal, vol. 19, no. 18, pp. 7822-7828, 15 Sept.15, 2019. doi: 10.1109/JSEN.2019.2920235 DOI 10.1109/jsen.2019.2920235 Publisher IEEE-INST ELECTRICAL ELECTRONICS ENGINEERS INC Journal IEEE SENSORS JOURNAL Rights © 2019 IEEE. Download date 25/09/2021 12:19:07 Item License http://rightsstatements.org/vocab/InC/1.0/ Version Final accepted manuscript Link to Item http://hdl.handle.net/10150/634549 > REPLACE THIS LINE WITH YOUR PAPER IDENTIFICATION NUMBER (DOUBLE-CLICK HERE TO EDIT) < 1 Simplified White Blood Cell Differential: An Inexpensive, Smartphone- and Paper-Based Blood Cell Count Matthew V. Bills, Brandon T. Nguyen, and Jeong-Yeol Yoon or trained lab specialist to prepare blood smear slides, stain Abstract— Sorting and measuring blood by cell type is them, and then manually count different WBC types using a extremely valuable clinically and provides physicians with key hemocytometer under a microscope [3]. To do this they must information for diagnosing many different disease states dilute specimens in a red blood cell (RBC) lysing solution to including: leukemia, autoimmune disorders, bacterial infections, remove RBCs and count WBCs. Manually counting WBCs is etc. Despite the value, the present methods are unnecessarily laborious and requires specialized medical equipment and costly and inhibitive particularly in resource poor settings, as they require multiple steps of reagent and/or dye additions and trained personnel. -
399808 AN-Metabolic Activity During MSC Differentiation Layv3
Automated monitoring of metabolic activity and differentiation of human mesenchymal stem cells Application Note REAL-TIME MEASUREMENTS WITH THE SPARK® MULTIMODE READER 2 INTRODUCTION Differentiation of MSC was monitored for up to 60 hours. Detailed analysis of human stem cells and the cellular Cells were seeded into the microplate and left to adhere processes underlying differentiation of these cells towards over night. The plate was then placed into the Humidity specialized cell types offers the potential for the specific Cassette, with the fluid reservoirs of the plate filled with and targeted treatment of diseases by cell-based deionized water. To avoid edge effects due to evaporation, therapies, also called regenerative medicine. the outer wells of the plate were filled only with culture Human mesenchymal stem cells (MSC) are multipotent medium without cells. The Spark 10M was set to 37°C and adult stem cells with a high capacity for self-renewal 5% CO2. Cell differentiation was monitored by automated originating from perivascular progenitor cells [1]. Using measurement of absorbance, cell confluence, appropriate differentiation media, MSC can be luminescence or fluorescence at defined intervals. differentiated in vitro towards adipocytes, chondrocytes, neurons and osteoblasts [2]. Reagents and measurement The Fluorometric Cell Viability Kit I (Resazurin; PromoCell, This application note describes the results of a study PK-CA707-30025-0) was used for viability assessment. aimed at identifying phenotypic changes during MSC The assay is based on the conversion of resazurin to its differentiation by automated, real-time analysis using the fluorescent product resorufin by a chemical reduction in Spark 10M multimode reader and several well-established living cells. -
Quantitative Phase Imaging in Biomedicine
REVIEW ARTICLE https://doi.org/10.1038/s41566-018-0253-x Quantitative phase imaging in biomedicine YongKeun Park1,2,3, Christian Depeursinge4,5 and Gabriel Popescu 6* Quantitative phase imaging (QPI) has emerged as a valuable method for investigating cells and tissues. QPI operates on unla- belled specimens and, as such, is complementary to established fluorescence microscopy, exhibiting lower phototoxicity and no photobleaching. As the images represent quantitative maps of optical path length delays introduced by the specimen, QPI provides an objective measure of morphology and dynamics, free of variability due to contrast agents. Owing to the tremen- dous progress witnessed especially in the past 10–15 years, a number of technologies have become sufficiently reliable and translated to biomedical laboratories. Commercialization efforts are under way and, as a result, the QPI field is now transi- tioning from a technology-development-driven to an application-focused field. In this Review, we aim to provide a critical and objective overview of this dynamic research field by presenting the scientific context, main principles of operation and current biomedical applications. maging biological cells and tissues is central to biological research will wash out the image altogether due to multiple scattering. Both and medical diagnosis. Following its four-century-old history, of these extremes result in poor image contrast. Single cells and thin microscopy has become the most commonly used tool in medi- tissue slices belong to the first category: the scattered light they gen- I 1 cine and biology . However, despite significant breakthroughs, opti- erate is orders of magnitude weaker than the incident light. -
Absolute Counting Using Flow Cytometry Bright Count
Absolute counting using flow cytometry Bright Count Microspheres ™ Single-platform system for absolute counts I PRODUCT MANUAL [REF] IQP-391 s 50 tests [REF] IQP-392 s 100 tests [IVD] In Vitro Diagnostic medical device IQ Products BV Rozenburglaan 13a 9727 DL Groningen, The Netherlands ENG IQP-391 – IQP-392 version 3 www.iqproducts.nl Bright Count Microspheres ™ Double internal standard for absolute counting by flow cytometry Bright Count Microspheres ™ [IVD] [REF] IQP-391 s 50 tests [IVD] [REF] IQP-392 s 100 tests This product is registered as “In Vitro Diagnostic medical device” in the countries that belong to the European Community. Intended use Bright Count Microspheres™ is designed for determining absolute counts of cells in peripheral blood, bone marrow, leukapheresis and culture medium samples using flow cytometry. Bright Count Microspheres™ is a microbead-based single-platform system for absolute counts, which can be used in combination with monoclonal antibodies conjugated with different fluorochromes, which make it possible to identify the cell subpopulations for which the absolute count is intended. Background In recent years, the enumeration of absolute levels of cells and their subsets has been shown to be relevant in different research settings as well as for clinical diagnosis laboratories. Absolute counting of cells or cell subsets by flow cytometry is an established technique in the enumeration of CD4 + and CD8 + T-lymphocytes for monitoring of patients with human immunodeficiency virus (HIV +) infection, in the enumeration of CD34 + haematopoietic stem and progenitor cells in patients who are candidates for bone marrow autotransplantation, and in the enumeration of residual leukocytes as part of the quality control of leukoreduced blood products (1,2,3) .