Abstract
The ability to accurately measure cell viability is important for any cell-based assay. Traditionally, viability measurements have been performed using the trypan blue exclusion method on a hemacytometer, which allows researchers to visually distinguish viable from nonviable cells. While the trypan blue method can work for cell lines or primary cells that have been rigorously purified, in more complex samples such as PBMCs, bone marrow, whole blood, or any sample with low viability, this method can lead to errors. In recent years, advances in optics and fluorescent dyes have led to the development of automated benchtop image-based cell counters for rapid cell concentration and viability measurement. In this work, we demonstrate the use of image-based cytometry for cell viability detection using single-, dual-, or multi-stain techniques. Single-staining methods using nucleic acid stains such as EB, PI, 7-AAD, DAPI, SYTOX Green, and SYTOX Red, and enzymatic stains such as CFDA and Calcein AM, were performed. Dual-staining methods using AO/PI, CFDA/PI, Calcein AM/PI, Hoechst/PI, Hoechst/DRAQ7, and DRAQ5/DAPI that enumerate viable and nonviable cells were also performed. Finally, Hoechst/Calcein AM/PI was used for a multi-staining method. Fluorescent viability staining allows exclusion of cellular debris and nonnucleated cells from analysis, which can eliminate the need to perform purification steps during sample preparation and improve efficiency. Image cytometers increase speed and throughput, capture images for visual confirmation of results, and can greatly simplify cell count and viability measurements.
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References
Cook JA, Mitchell JB (1989) Viability measurements in mammalian cell systems. Anal Biochem 179:1–7
Oh H, Livingston R, Smith K et al (2004) Comparative study of the time dependency of cell death assays. MURJ 11:53–62
Chan LL, Zhong XM, Qiu J et al (2011) Cellometer vision as an alternative to flow cytometry for cell cycle analysis, mitochondrial potential, and immunophenotyping. Cytometry Part A 79A:507–517
Chan LL, Wilkinson AR, Paradis BD et al (2012) Rapid image-based cytometry for comparison of fluorescent viability staining methods. J Fluoresc 22:1301–1311
Saldi S, Driscoll D, Kuksin D et al (2014) Image-based cytometric analysis of fluorescent viability and vitality staining methods for ale and lager fermentation yeast. J Am Soc Brew Chem 72:253–260
Han X, Liu Z, Mc J et al (2015) CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation. Sci Adv 1:1–8
Shah D, Naciri M, Clee P et al (2006) NucleoCounter—an efficient technique for the determination of cell number and viability in animal cell culture processes. Cytotechnology 51:39–44
Al-Rubeai M, Welzenbach K, Lloyd DR et al (1997) A rapid method for evaluation of cell number and viability by flow cytometry. Cytotechnology 24:161–168
Strober W (2001) Monitoring cell growth. In: Current protocols in immunology, vol APPENDIX 3A
Shapiro HM (2004) “Cellular Astronomy” - a foreseeable future in cytometry. Cytometry Part A 60A:115–124
Stoddart M (2011) Cell viability assays: introduction. Methods Mol Biol 740:1–6
Davey HM, Kell DB (1996) Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. Microbiol Rev 60:641–696
Michelson AD (1996) Flow cytometry: a clinical test of platelet function. Blood 87:4925–4936
Tibbe AGJ, de Grooth BG, Greve J et al (2002) Imaging technique implemented in CellTracks system. Cytometry Part A 47:248–255
Shapiro HM, Perlmutter NG (2006) Personal cytometers: slow flow or no flow? Cytometry Part A 69A:620–630
Gerstner AOH, Mittag A, Laffers W et al (2006) Comparison of immunophenotyping by slide-based cytometry and by flow cytometry. J Immunol Methods 311:130–138
Mital J, Schwarz J, Taatjes DJ et al (2005) Laser scanning cytometer-based assays for measuring host cell attachment and invasion by the human pathogen Toxplasma gondii. Cytometry Part A 69A:13–19
Hall A, Wu L-P, Parhamifar L et al (2015) Differential modulation of cellular bioenergetics by poly(l-lysine)s of different molecular weights. Biomacromolecules 16:2119–2126
Siqueira-Neto JL, Moon S, Jang J et al (2012) An image-based high-content screening assay for compounds targeting intracellular Leishmania donovani amastigotes in human macrophages. PLoS Negl Trop Dis 6:e1671
Zanella F, Lorens JB, Link W (2010) High content screening: seeing is believing. Trends Biotechnol 28:237–245
Schepers K, Pietras EM, Reynaud D et al (2013) Myeloproliferative neoplasia remodels the endosteal bone marrow niche into a self-reinforcing leukemic niche. Cell Stem Cell 13:285–299
Szabo SE, Monroe SL, Fiorino S et al (2004) Evaluation of an automated instrument for viability and concentration measurements of cryopreserved hematopoietic cells. Lab Hematol 10:109–111
Macfarlane RG, Payne AM-M, Poole JCF et al (1959) An automatic apparatus for counting red blood cells. Br J Haemacytol 5:1–15
Verso ML (1971) Some nineteenth-century pioneers of haematology. Med Hist 15:55–67
Falzone N, Huyser C, Franken D (2010) Comparison between propidium iodide and 7-amino-actinomycin-D for viability assessment during flow cytometric analyses of the human sperm acrosome. Andrologia 42:20–26
Gordon KM, Duckett L, Daul B et al (2003) A simple method for detecting up to five immunofluorescent parameters together with DNA staining for cell cycle or viability on a benchtop flow cytometer. J Immunol Methods 275:113–121
Jarnagin JL, Luchsinger DW (1980) The use of fluorescein diacetate and ethidium bromide as a stain for evaluating viability of mycobacteria. Biotech Histochem 55:253–258
Roth B, Poot M, Yue S et al (1997) Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain. Appl Environ Microbiol 63:2421–2431
Wlodkowic D, Skommer J, Faley S et al (2009) Dynamic analysis of apoptosis using cyanine SYTO probes: from classical to microfluidic cytometry. Exp Cell Res 315:1706–1714
Bratosin D, Mitrofan L, Palii C et al (2005) Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging. Cytometry Part A 66A:78–84
Jones KH, Senft JA (1985) An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide. J Histochem Cytochem 33:77–79
Donoghue AM, Garner DL, Donoghue DJ et al (1995) Viability assessment of Turkey sperm using fluorescent staining and flow cytometry. Poult Sci 74:1191–1200
Mascotti K, McCullough J, Burger SR (2000) HPC viability measurement: trypan blue versus acridine orange and propidium iodide. Transfusion 40:693–696
Cai K, Yang J, Guan M et al (2005) Single UV excitation of Hoechst 33342 and propidium iodide for viability assessment of rhesus monkey spermatozoa using flow cytometry. Arch Androl 51:371–383
Smith PJ, Wiltshire M, Davies S et al (1999) A novel cell permeant and far red-fluorescing DNA probe, DRAQ5, for blood cell discrimination by flow cytometry. J Immunol Methods 229:131–139
Akagi J, Kordon M, Zhao H et al (2013) Real-time cell viability assays using a new anthracycline derivative DRAQ7®. Cytometry Part A 83A:227–234
Sutkeviciene N, Andersson MA, Zilinskas H et al (2005) Assessment of boar semen quality in relation to fertility with special reference to methanol stress. Theriogenology 63:739–747
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Chan, L.LY., McCulley, K.J., Kessel, S.L. (2017). Assessment of Cell Viability with Single-, Dual-, and Multi-Staining Methods Using Image Cytometry. In: Gilbert, D., Friedrich, O. (eds) Cell Viability Assays. Methods in Molecular Biology, vol 1601. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6960-9_3
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DOI: https://doi.org/10.1007/978-1-4939-6960-9_3
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