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Comparison of Trypan Blue Dye Exclusion and Fluorometric Assays for Mammalian Cell Viability Determinations Steven A. Altman? Lisa Randers? and Govind Rao**t** Department of Chemical and Biochemical Engineering, College of Engineering, University of Maryland Baltimore County, Baltimore, Maryland 21228, and Medical Biotechnology Center of the Maryland Biotechnology Institute, University of Maryland, Baltimore, Maryland 21201
A hemocytometer-based trypan blue dye exclusion cell quantitation and viability assay was compared with a similar assay using simultaneous fluorometric staining with fluorescein diacetate and propidium iodide. Viable and nonviable cell densities were measured, and culture viability was calculated both during the normal growth cycle of a murine hybridoma and in response to the application of millimolar concentrations of either tert-butyl hydroperoxide or ferrous iron. During the early phase of rapid hybridoma cell growth, assay-based differences in viable cell density were not significant. As the culture aged, the trypan blue dye exclusion assay significantly overestimated cell viability, thereby underestimating nonviable cell density and yielding an erroneous estimation of the overall viability of the culture. Because of ita lack of ambiguity in the identification of stained, nonviable cella and ita resulting increased accuracy in the estimation of culture viability, the fluorometric assay was considered a better choice for the evaluation of cell viability.
Introduction Rapid, accurate estimation of cell viability is vital to successful mammalian cell and tissue culture, to bioreactor design evaluation and scale-up procedures, to evaluation of cytotoxic or toxicological properties of compounds used experimentally, and to the production or recovery of exogenous compounds by transformed cells. The trypan blue dye exclusion assay is the most commonly utilized test for cell viability (Mishell and Shiigi, 1980). The usefulness of this procedure is limited since the number of blue-staining cells increases following addition of the dye, requiring that cells be counted within 3-5 min (Hudson and Hay, 1980). Additionally, trypan blue is reported to generally overestimate cell viability (Smith and Smith, 19891, to yield inaccurate results following trypsinization, EDTA treatment, or scraping of anchored cells (Tennant, 1964), and to be excluded from both metabolically viable and nonviable (senescent or dead) cells when used in cell culture media containing serum proteins (Black and Berenbaum, 1964). Numerous reporta have appeared suggesting that fluorescent dyes are more accurate and reliable indicators of cell viability (Edidin, 1970; Persidsky and Baillie, 1977). Because fluorescent dyes are stable following uptake by cells, viability may be determined several days.following application (Jones and Senft, 1985). Nonspecificcleavage of fluoresceindiacetate by esterasesin metabolicallyviable cells is well characterized (Rotmanand Papermaster, 1966). Fluorescein diacetate cleavage results in the formation of fluorescein, a compound which fluoresces green in metabolically active, viable cells. Similar to trypan blue, propidium iodide and other DNA-intercalating dyes estimate cell membrane transport properties. Such dyes are reportedly taken up by cells that have lost their membrane permeability barrier or dye exclusion capacity Author to whom correspondence should be addressed. ‘Department of Chemical and Biochemical Engineering. Medical Biotechnology Center of the Maryland Biotechnology Institute.
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87567938/93/3009-0671$04.00/0
and are thus considered nonviable (Crissman et al., 1979). The intensity of orange fluorescenceobserved in nonviable cells may depend on the extent of nuclear membrane disruption. Intercalating dyes are therefore well suited for identifyingnonviable cells. The current work comparea trypan blue dye exclusion and a modified fluorometric cell viability assay (Jones and Senft, 1985)both for routine use in cell culture and for evaluating the cytotoxicity of compounds applied experimentally to live cells.
Materials and Methods Cells. Suspension-cultured SPa/O-derivedmurine hybridomas, HyHEL-10 (courtesy of S. J. Smith-Gill, National Cancer Institute, National Institutes of Health, Bethesda, MD), were amplified in Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 (DME/F-12)supplemented with L-glutamine,50 mg/L gentamicin, 2-mercapt~ethano1, NaHC03, and 4% (v/v) fetal bovine serum. Cells were maintained at 37 OC in a 5 % COz atmosphere and were subcultured in the mid-exponential growth phase. For experimental use, cultures were grown to a density of approximately(7.0-8.0) X lo5viable cells/&, equivalent to approximately 8.0 X lo5-1.0 X lo6 total cells/&. Cell Viability Determination. For cell growth cycle viability studies, a uniform suspension of cells was inoculated into triplicate 75 cm2tissue culture flasks and maintained in darkness in a standard COZ incubation chamber. Replicate samples from all flasks were counted each day for 6 days using both dye exclusion and fluorometric assays of cell viability. Comparative cell viability stain experiments were conducted three times. Following analysis of variance, data from all experiments were pooled for further statistical analysis. For trypan blue staining, 200 pL of cells was aseptically transferred to a 1.5-mL clear Eppendorf tube and incubated for 3min at room temperature with an equal volume of 0.4% (w/v)trypan blue solution prepared in 0.81 % NaCl and 0.06 % (w/v)dibasic potassiumphosphate. Cells were counted using a dual-chamberhemocytometer and a light microscope. Viable and nonviable cells were recorded
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Figure 1. Change in cell density during the growth cycle of HyHEL-10 murine hybridoma cells. Viable (solid symbols) and nonviable cells (open symbols) were identified by either trypan blue dye exclusion (0,o)or simultaneous fluorescein diacetate/ propidium iodide (m, 0 ) viability assays.
Figure 2. Change in suspension culture viability during the growth cycle of HyHEL-10 murine hybridoma cella calculated on the basis of resulta obtained by either trypan blue dye exclusion (0) or simultaneous fluorescein diacetate/propidium iodide ( 0 ) viability assays.
separately,and the means of three independent cell counts were pooled for analysis. Fluorescein diacetate and propidium iodide fluorescent stains were prepared for cell viability determination as previously described (Jones and Senft, 1985). Approximately 100pL of cells aseptically transferred to a 1.5-mL amber Eppendorf tube were incubated for 10 s in 100 p L of fluorescein diacetate solution and 30 pL of prepared propidium iodide solution. Stained cells were counted in a separate dual-chamber hemocytometerusing a standard fluorescencemicroscope equipped with a 100-Wmercuryhalogen light source and a wide-band (435-490)interference blue excitation dichroic band passlbarrier filter cube (OlympusOptical Co. Ltd., Tokyo) that permitted simultaneous viewing of both live and dead cells. Cell toxicity studies were conducted on replicate 75 cm2 flasks of cells in the late phase of rapid cell growth. Cells were held in a 37 "C shaking water bath and were equilibrated in air for 30 min prior to the application of varying concentrations of either ferrous iron [Fe(II)I or tert-butyl hydroperoxide (t-BuOOH). At 15-minintervals, 0.5 mL of cell suspension was removed from each flask for the viability determination. In all instances, both cell viability assays were conducted simultaneously. Toxicity evaluation experiments were conducted three times, and data from allexperimentswere pooled for further statistical analysis.
Early in the growth cycle during the phase of rapid cell growth, no significant differences in blue-stained (nonviable cells, trypan assay) or orange fluorescent(nonviable cells, fluorometric assay) cell number (Figure 1,days 1-2) or in cell culture viability (Figure 2) were observed. As the number of nonviable cells began to increase following the phase of slow cell growth, assay-based differences in the estimation of nonviable cell number were significant. Fewer nonviable cells were detected using trypan blue dye exclusion (Figure 1, days 3-6). The resulting estimation of culture viability (Figure 2, days 3-6) was therefore higher when calculatedon the basis of cell counts obtained using this assay method. On the basis of cell counts obtained using the fluorometric viability assay, approximately 52 % of the cells in the culture were viable at the end of the experiment, whereas culture viability was estimated at approximately 70% on the basis of cell counts obtained using the trypan blue dye exclusion assay (Figure 2, day 6). The overestimation of cell viability by trypan blue may be due, in part, to its insensitivity to nonviable cells, to the subjective nature of the assay, and to the requirement that cell counts be made rapidly followingaddition of the dye. Since dying nonviable cells may partially exclude the dye, such cells may appear unstained and thus be counted as viable. Dead hybridoma cells may lose considerable volume and be disregarded as debris. Cell nucleiand celldebris are also stained by trypan blue, which may further compromise accuracy. Additionally, since the assays were conducted on cells in media containing serum, the formation of serum protein-trypan blue complexeswhich are unable to diffuse into nonviable cells cannot be discounted as a source of error (Black and Berenbaum, 1964). CytotoxicityEvaluation. Experiments involving the treatment of cells with exogenous compounds should include an assessment of the toxicity of the compounds to properly evaluate their base-line metabolic effects on the cell. During experiments designed to demonstrate free radical mediated damage to mammalian DNA (Dizdaroglu et al., 19911,intact hybridoma cells were treated with Fe(I1) and t-BuOOH to elucidate their mechanism and chemistry of damage. It was important to evaluate the cytotoxicityof these compoundsin order to accurately evaluate the resulting pattern of DNA damage. No decrease in cell viability was observed in cultures treated with 0.1 mM t-BuOOH using either dye exclusion or fluorometric methods (Figure 3). At a concentration of 1.0 mM, decreased viability was detected using the
Results and Discussion Growth Cycle. Cellviability was evaluatedover a 6-day growth cycle using both colorimetric and fluorometric assays. Hemocytometer-basedcounts of totalcell number (viableplus nonviablecells)obtained by the two techniques differed by an overall mean of 6.06% f 1.51 % standard error. Cells were normally subcultured every 40 h by transferring 2.0 mL of cells into 10mL of fresh media. The low initial number of both stained and unstained cells (Figure 1, day 0) was due to the dilution effect of transfer into fresh media. A plot of unstained (viablecells,trypan assay) or green fluorescent (viable cells, fluorometric assay) cell density yielded similar growth curves (Figure 1,days 1-31) although differences in cell density were observed throughout the cell growth cycle. Assay-based differencesin viable cell number were more pronouncedlate in the growth cycle (Figure 1,days 4-6)as the overall viability of the culture declined.
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Figure 3. Change in nonviable cell density (solid symbols) and calculated culture viability (open symbols) as determined by trypan blue dye exclusion (A) or simultaneous fluorescein diacetate/propidium iodide (B) viability assays in response to treatment of HyHEL-10 murine hybridoma cells wlth 0 (m, O ) , 0.1 (0, 0 ) or 1.0 mM (A,A) tert-butyl hydroperoxide. Solid symbols map to the left and open symbols to the right ordinate axis.
Figure 4. Change in nonviable cell density (solid symbols) and calculated culture viability (open symbols) as determined by trypan blue dye exclusion (A) or simultaneous fluorescein diacetate/propidium iodide (B)viability assaye in response to treatment of HyHEL-10 murine hybridoma cells with 0 (m, c]), 0.01 (0,0),0.1 (A,A), or 1.0 mM (e, 0 ) ferrous iron. Solid symbols map to the left and open symbols to the right ordinate axis.
fluorometric assay after 45 min of exposure, whereas no significant decrease in viability was observed in cultures counted using the dye exclusion assay until 75 min following treatment. As the number of dead cells detected using the fluorometric assay method increased, the estimation of culture viability based on cell counts obtained using the dye exclusion assay became more erratic. On the basis of results obtained using trypan dye exclusion, the viability of cultures treated with 1.0 mM t-BuOOH appeared to be greater than that of untreated control cultures at 45-60 min following treatment and increased at 105 min following a significant decline in viability detected at 90 min (Figure 3). No significant increase in nonviable cell density and no decrease in culture viability were detected using the trypan dye exclusion assay in cultures treated with any of four concentrations of Fe(I1) (Figure 4). In contrast, following approximately 60 min of treatment with 0.1 or 1.0 mM Fe(II), a 5% decrease in culture viability was observed in cultures counted using the fluorometric assay. The slow accumulation of nonviable cells detected by the fluorometric assay resulted in a 10% decrease in viability during the 120-minperiod of observation (Figure 4). The ability to detect such subtle changes in viability using the fluorometric viability assay was enhanced by equipping the fluorescence microscope with a wide-band (550-800) interference green excitation dichroic band paselbarrier filter cube that permitted the observation of only orange fluorescent cells. This permitted the detection of cells which took up and intercalated propidium iodide into nucleic acids still retained by the intact nuclear membrane or in which other components of nonviable cells were additionally stained. Such cells initially appeared unstained when viewed with the wide-band blue filter cube. However,when viewed with the alternatewide-band green
filter cube, a tiny orange dot of high fluorescent intensity was evident.
Conclusion In addition to documented interferences aseociated with the trypan blue dye exclusion viability assay, the assignment of trypan-stained cells to viable or nonviable categories was found to be subjective and arbitrary. The fluorometric assay is also subject to interferences, in that fluorescein diacetate may be converted to fluorescein by residual esterase activity in nonviable cells. Likewise, in addition to nucleic acids, propidium iodide may bind to proteins and glycosaminoglycanson the plasma membrane of viable cells. However, in the current work fluorescein diacetate and propidium iodide fluorescent dyes were applied simultaneously, so that all cells fluoresced either orange or green. The high fluorescent intensity of each dye tended to mask any opposite fluorescence caused by artifacts, thereby eliminating any ambiguity in the identification of stained cells. Since the fluorescence microscope used in this study permitted rapid switching among multiple dichroic filters, the fluorescent status of any cell could be immediately confirmed by viewing with an alternate filter. Therefore, the unambiguous identification of fluorescent status and the resulting accuracy in the estimation of culture viability made the fluorometric assay more suitable both for routine use in cell culture and for accurate compound cytotoxicity evaluation.
Acknowledgments Funding from NIH Grant RR06562 and NSF Grant BCS 9157852, with matching funds from Artisan Industries, Inc., Waltham, MA, is acknowledged. We thankDr. Simon Kwong for valuable assistance with technical suggestions and manuscript preparation and Marwan Akar for technical support.
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Literature Cited Black, L.; Berenbaum, M. C. Factors Affecting the Dye Exclusion Test for Cell Viability. Exp. Cell Res. 1964,35, 9-12. Crissman, H.; Stevenson, A.; Kissane, R.; Tobey, R. In Flow Cytometry clnd Sorting; Melamed, M., Mullaney, P., Mendelsohn, M., Eds.; John Wiley and Sons: New York, 1979;pp 243-261. Dizdaroglu, M.; Nackerdien, Z.; Chao, B.-C.; Gajewski, E.; Rao, G. ChemicalNature of in vivo DNA Base Damage in Hydrogen Peroxide-TreatedMammalian Cells. Arch. Biochem. Biophys. 1991,285,388-390. Edidin, M. A Rapid, Quantitative Fluorescence Assay for Cell Damage by Cytotoxic Antibodies. J. Immunol. 1970, 104, 1303-1309. Hudson, L.; Hay, F. C. In Practical Immunology; Blackford Scientific Publications: Oxford, U. K., 1980; pp 29-31. Jones, K. H.; Senft, J. A. An Improved Method to Determine Cell Viability by Simultaneous Staining with Fluorescein Diacetate-Propidium Iodide. J.Histochem. Cytochem. 1985, 33,77-79.
Mishell, B. B.;Shiigi, S. M. In Selected Methods in Cellular Immunology; Freeman and Co.: San Francisco, 1980;pp 1619. Peraidsky, M. D.; Baillie, G. S. Fluorometric Test of Cell Membrane Integrity. Cryobiology 1977,14,322-326. Rotman, B.;Papermastar, B. W. Membrane Properties of Living Mammalian Cells as Studied by Enzymatic Hydrolysis of Fluorogenic Esters. Proc. Natl. Acad. Sci. U.S.A. 1966,55, 134-141. Smith, A. L.; Smith, H. V. A Comparisonof FluoresceinDiacetate and Propidium Iodide Staining And in vitro Excystation for Determining Giardia intestinalis Cyst Viability. Parmitology 1989,99,329-331. Tennant, J. R. Evaluation of the Trypan Blue Technique For Determination of Cell Viability. Transplantation 1964,2,686 689. Accepted July 29,1993.’ ~
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Abstract published in Advance ACS Abstracts, October 1,1993.
