Bbtechnol. Rag. 1999, 9, 871-874
671
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.
*
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
0 1993 American Ctmmlcal Society and American Institute of Chemlcal Engineera
672
Biotechnol. Prog, 1993, Vol. 9, No. 6
2.5 IO6
T
100
80
70
DayO
Day 1
Day2
Day3
Day4
Day5
Day6
DayO
Day 1
Day2
Day3
Day4
Day5
Day6
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.
Wiotechnoi.Pyog., 1993. Vol. 9, No. 6
674
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Accepted July 29,1993.’
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Abstract published in Advance ACS Abstracts, October 1,1993.