Determining cell viability is a vital component in many biological experiments and one of the most common methods for this measurement is the trypan blue (TB) exclusion assay. Previous studies have shown that TB exclusion assays reported significantly higher viabilities when compared to fluorescence-based methods, but these studies have not provided evidence on potential reasons for the differences.
Download this whitepaper to learn how an image-based cytometry method was used to investigate:
- The effectiveness of TB in samples with low viability
- How light microscopy may affect results
- The differences between room temperature-incubated and heat-shocked cells
Comparison of Trypan Blue and
Fluorescence-Based Viability Detection
Methods Via Morphological
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1001253 Rev. A
Comparison of Trypan Blue and Fluorescence-Based
Viability Detection Methods Via Morphological
Determining cell viability is a vital component in many biological experiments that range
from standard cell culture to the investigation of pharmacological agents on tumor cells. One
of the earliest and most common methods for measuring cell viability is the trypan blue (TB)
exclusion assay [1, 2]. Over the last two decades, there have been various publications on
comparing TB exclusion and fluorescence-based cellular viability assays [1, 3-5]. Previous
results have shown that in a time-course measurement, TB exclusion assays reported
significantly higher viabilities when compared to fluorescence-based methods [4, 5]. These
previous studies have only shown comparison results, but did not provide evidence on
potential reasons for the differences. In this work, we performed a time-course study to
compare cellular viability measured with TB and AO/PI using an automated image-based
cytometry method (Cellometer, Nexcelom Bioscience).
Materials and Methods
Jurkat cell preparation
Jurkat cells were cultured in RPMI-1640 Medium. For a viability comparison of room
temperature-incubated cells, one flask was taken out of the incubator and placed into a
bench-top drawer. The flask was kept at room temperature for the remainder of the
experiment. A small aliquot of cells was removed from the flask at the following time points:
0, 6, 12, 24, 48, 72, 96, and 168 h. A similar experiment was conducted to study the effect of
different TB concentrations on Jurkat cell viability, which was measured at the following
time points: 0, 3, 6, 9, 12, 27, and 33 h. (The time points were shorter than the first
experiment because it was targeted towards the time frame where the viability difference,
shown here in Figure 2, was most apparent.) For a heat shock viability comparison, Jurkat
cells were placed in boiling water for 15 min. After heat shock, five samples were prepared at
viabilities of 0, 25, 50, 75, and 100% by mixing the heat-killed and fresh Jurkat cells at the
Time-course viability of room temperature-incubated Jurkat cells
The viability and concentration of the room temperature-incubated Jurkat cells were
determined and compared using four detection methods: (1, 2) measuring viability staining
with PI or AO/PI and counting with Cellometer Vision, (3) staining with 0.4% TB and
counting with Cellometer AutoT4, and (4) staining with 0.4% TB and manual counting with
hemacytometer. The AO/PI staining procedure was performed as previously described .
Room temperature-incubated cell samples at 0, 6, 12, 24, 48, 72, 96, and 168 h were mixed
with PI, AO/PI, or TB, and analyzed immediately with Cellometer Vision, AutoT4, and
hemacytometer. The viability and concentration were measured in quadruplicate for all time
Trypan blue concentration-dependent viability comparison
The effect of TB concentration on viability of room temperature-incubated Jurkat cells was
determined and compared using two detection methods: (1) measuring viability by AO/PI
and counting with Cellometer Vision, and (2) staining with various concentrations of TB and
manual counting with hemacytometer. Each room temperature-incubated cell sample at 0, 3,
6, 9, 12, 27, and 33 h was mixed with AO/PI or 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125% of TB and
analyzed immediately with Cellometer Vision and hemacytometer. The viability and
concentration were measured in quadruplicate for all time points.
Heat shock viability comparison
Freshly collected Jurkat cells were separated into two equal aliquots, one was heat shocked
by boiling the samples for 15 min (100% non-viable) and the other was not (100% viable).
The cells from these two aliquots were then mixed at different ratios to produce cell
suspensions. The viability and concentration of each heat-shocked/live viability mixture (0,
25, 50, 75 and 100%) was determined and compared using three detection methods: (1)
Measuring viability staining with PI and counting with Cellometer Vision, (2) staining with
0.4% TB and counting with Cellometer AutoT4, and (3) staining with 0.4% TB and manual
counting with hemacytometer. Each heat-shock/live mixture sample was mixed with PI or
TB, and analyzed immediately with image cytometer and hemacytometer. The viability and
concentration for each of the three independent experiments were measured in quadruplicate
for all five mixtures.
Image cytometry automated cell-counting protocol
Jurkat cells stained with PI, AO/PI, or TB were pipetted immediately after mixing into a
Nexcelom disposable counting chamber. The Cellometer Vision utilizes fluorescence (FL)
optics modules of VB-535- 402 (pseudo-color green) and VB-660-502 (pseudo- color red) to
detect AO and PI, respectively. The Cellometer AutoT4 uses a color camera to detect TB
stained Jurkat cells. For TB image analysis, live cells with bright center and dark cells
stained with TB in the brightfield (BF) were enumerated. For PI image analysis, total cells in
the BF and dead cells in the FL channel were enumerated. For AO/PI image analysis, AOstained live cells and PI-stained dead cells in green and red FL channels were enumerated.
Hemacytometer manual cell counting protocol
Jurkat cells stained with TB were pipetted immediately into a Neubauer hemacytometer. Live
and dead cells were enumerated manually under light microscopy for samples from both
controlled heat-shocked and room temperature-incubated Jurkat cells. Cell samples with an
approximate 50% viability were used for manual counting.
Figure 1. Time-course viability of room temperature-incubated Jurkat cells
• BF images of AO/PI and TB stained room temperature-incubated Jurkat cells.
Samples were measured at time points 0 - 168 h post incubation. Large, dim, and
diffused cells in the BF images are shown with red and green arrows, respectively.
• Cells from 0 to 12 h resembled those that were heat-shocked; outer cell membranes
appeared to be dark and well defined. By 168 h, cells now appeared grayish and
undefined. Similar morphological characteristics were observed, where cell
membranes changed from well-defined (tight and darkly stained) to amorphous (large
• While the number of TB-stained large, dim cells increased with time, the
morphological characteristics of Jurkat cells stained with AO did not change over the
same period of time. We visually confirmed that the number of stained (AO/PI and
TB) dead Jurkat cells increased over time.
• The most optimal method for measuring cell viability is the AO/PI method, which
allows for dual-fluorescence detection (live and dead), thus eliminating the potential
inaccurate counting of membrane-poor cells populations: late apoptotic and necrotic
cells, as well as eliminating the counting of cellular debris.
Figure 2. Concentration and viability time-course measurement of TB, PI, and
AO/PI-stained room temperature-incubated Jurkat cells
• (a) The four detection methods showed clear differences between fluorescence-based
and TB exclusion assays.
o A two-sample T-test was calculated at 12 h time point comparing AO/PI to PI
and AO/PI to TB staining. The results showed that AO/PI was comparable to
PI staining method (p > 0.05), while AO/PI was significantly different from
TB counting (p ≤ 0.05).
o At the 24 h time point, the viability measured by TB showed approximately
80%, while the AO/PI or PI showed 70%.
• (b) The total concentrations were also measured by AO/PI and PI methods, which
showed consistent results, whereas the TB method showed a gradual decrease over
• (c) The live cell concentrations were measured and showed no differences between
the two methods, however the dead cells showed higher concentrations for AO/PI and
PI methods than the TB method over time.
• By the end of the time trial, both the manual and TB measured concentrations were
more than two times lower than the fluorescence-based detection methods.
Furthermore, the initial deviation in the number of measured dead cells by TB began
at 12 h post incubation at room temperature which is the same time at which the
measured cell viability decreased to ~70% by AO/PI. These data suggest that as the
cell viability decreases, the TB exclusion method undercounts the number of dead
cells thereby producing an inflated viability measurement.
Figures 3 and 4. Trypan blue concentration dependence viability comparison
• (3) In addition to the increase of large, dimly diffused shaped cells over time (0-33 h
post incubation; red arrows), high concentrations of TB exhibited more of these cells
in comparison to lower concentrations.
• AO/PI staining remained consistent with the previous experiment, showing increase
in PI positive cells over the course of the experiment.
• (4a-c) As before, the viabilities measured from each TB concentration were higher
than the counts from AO/PI-stained cells. Similarly, the live cell concentrations were
comparable to each other when a two-sample T-test was calculated for AO/PI against
all TB concentrations (p > 0.05). The measured concentration of dead cells for each
experimental parameter was significantly different (p ≤ 0.05) for AO/PI against all
TB concentrations (b, c). And as in the previous time course experiment, even at high
TB concentrations, we observe a considerably lower number of reported dead cells.
• These data support not only our previous findings that TB viability measurement
overestimates measured sample viability percentage, but also show that this
phenomenon occurs at every tested TB concentration.
Figures 5 and 6. Viability comparison between heat-shocked and fresh Jurkat cells
• (5) BF and FL images of TB and PI-stained heat-shocked Jurkat cells at various
viability percentages. In these heat-shocked Jurkat samples, the TB images showed a
clear distinction between live and dead cells, where the dead cells exhibited a tight
dark and well-defined morphology. Similarly, the dead cells in the BF images
maintained an easily detectable cellular membrane.
• There were also clear morphological differences between the heat-shocked and room
temperature-incubated cells. Because the heat-shocked cells did not undergo natural
apoptotic cell death and necrosis, the stained Jurkat cells appear uniform in shape,
color, and roundness. In contrast, cells that were incubated at room temperature (seen
in Figure 3) exhibited non-uniform cellular morphology and non-uniform TB staining
• (6) Due to the morphological uniformity of heat-shocked cells, the viability counts
between TB and PI showed a high degree of correlation across the theoretical
viabilities. In this experiment, PI was used instead of AO/PI due to the lack of cellular
debris. In contrast, room temperature-incubated cells generated large amounts of
cellular debris. As a result, AO/ PI was required to accurately determine the viability
of nucleated cells in the sample without interference from the cellular debris .
Figure 7. Time course and percentages of dim, “balloon” cells versus dead, “dark”
cells at various TB concentrations
• Time-course concentration measurement of large dim cells (‘‘balloon’’) and dark
tight (dead) shaped Jurkat cells at TB concentrations. (a) 0.4%, (b) 0.1%, and (c)
0.025%. (d) Concentration results (at 33 h post incubation at room temperature)
showed that at higher TB concentration, the dim, diffuse cells or ‘‘balloons’’ were
very high in number compared those in the lowest TB concentration.
• The purpose of this study is to investigate the differences and possible causes of those
differences in viability measurements between the TB exclusion assay and
fluorescence-based viability stains. Over the years, there have been numerous
comparisons between the two viability detection methods, but the reports have not
proposed any possible explanations for the morphological changes caused by TB.
• The membranes of room temperature-incubated Jurkat cells underwent extreme
changes during cell death. In samples with high viability, the TB stained dead cells
exhibited tight dark uniform characteristics. This observation suggests that even
though the cells were permeable to TB, cellular membranes remained intact enough
to retain the TB dye in the cytosol.
• In samples with low viability, the TB stained dead cells exhibited large, dim, nonuniform diffused characteristics, which suggests functional loss of cell membrane
integrity. It appears that in those cells, TB was not contained within the cytosol,
which led to the formation of the observed large, dim cells.
• Because the dimmer cells may be difficult to count in hemacytometer under light
microscopy, the TB exclusion assay may artificially produce higher viability counts
due to the unintended exclusion of dead cells. As a result, the measured viability
between the two methods showed significant discrepancy, as high as 30%, where
similar observations have been made in previous publications .
• The morphological differences between room temperature-incubated and heatshocked cells can also potentially explain the variations in viability reporting of
comparison between TB and fluorescence-based methods.
• In addition, we examined the potential morphological changes that may be caused by
different concentrations of TB. Upon staining the cells with a higher concentration of
TB (0.4%), we observed a rapid formation of large dim diffuse circular shapes
(“balloons”; Fig. 9) that can be observed under light microscopy and Cellometer. The
balloons appear to vary in size and brightness, some more clearly diffuse than others.
• At lower TB concentrations (0.025%), we did not observe many balloon-like cells
(Fig. 9). At 0.025% TB, the concentration of the dye may be too low to adequately
stain all dead cells. Even though the nature of the balloons made them difficult to
count, the balloons were carefully identified and manually counted under a light
microscope across the concentrations of TB. We observed that at the 33 h time point,
for both 0.4 and 0.1% concentrations there are significantly higher number of diffused
balloon cells than the dark dead cells (p ≤ 0.05, for both TB concentrations). At
0.025% concentration there was not only a reversal in the number of counted balloons
versus the number of counted dead cells (p ≤ 0.05), but there was also a lower number
of total counted cells as well; which can be attributed to low dye concentration.
• Together these data suggest that TB, at high concentrations, has a morphological
effect on membrane compromised dead cells, thus producing balloon-like cells. At
lower concentrations of TB these dye-induced effects were not observed.
• Finally, because these dead cells appear as faint diffused colored balloon structures
they are very difficult to see and count, which leads to an overestimation of viability
measurement in the sample.
• Other potential reasons for the differences between TB and fluorescence-based
method can also be hypothesized. For example, PI has been shown to enter cells
earlier than TB, thus it has the potential to measure more dead cells. Also, since TB is
a cytoplasmic dye that stains intracellular proteins, dying cells with variation in
membrane degradation may not retain TB because of the loss of membrane integrity.
As a result, the number of dead cells counted may be lowered which in turn can
generate higher viability measurement. Meanwhile PI, an intercalating dye, binds to
the nucleic acid of the cells and is retained within the nucleus regardless of membrane
integrity. It is because of these properties that PI is routinely used to stain and identify
late apoptotic and necrotic cell populations [8, 9].
1. Altman, S.A., L. Randers, and G. Rao, Comparison of Trypan Blue Dye Exclusion and Fluorometric Assays
for Mammalian Cell Viability Determinations. Biotechnology Progress, 1993. 9: p. 671-674.
2. Louis, K.S. and A.C. Siegel, Cell Viability Analysis Using Trypan Blue: Manual and Automated Methods, in
Mammalian Cell Viability: Methods and Protocols, M.J. Stoddart, Editor. 2011, Springer. p. 7-12.
3. Black, L. and M.C. Berenbaum, Factors Affecting the Dye Exclusion Test for Cell Viability. Experimental Cell
Research, 1964. 35: p. 9-13.
4. Jones, K.H. and J.A. Senft, An Improved Method to Determine Cell Viability by Simultaneous Staining with
Fluorescein Diacetate-Propidium Iodide. Journal of Histochemistry and Cytochemistry, 1985. 33(1): p. 77-79.
5. Mascotti, K., J. McCullough, and S.R. Burger, HPC viability measurement: trypan blue versus acridine
orange and propidium iodide. Transfusion, 2000. 40: p. 693-696.
6. Bank, H.L., Assessment of islet cell viability using fluorescent dyes. Diabetologia, 1987. 30: p. 812-816.
7. Chan, L.L., et al., Accurate measurement of peripheral blood mononuclear cell concentration using image
cytometry to eliminate RBC-induced counting error. J Immunol Methods, 2013. 388(1-2): p. 25-32.
8. Denecker, G., et al., Phosphatidyl serine exposure during apoptosis precedes release of cytochrome c and
decrease in mitochondrial transmembrane potential. FEBS Letters, 2000. 465: p. 47-52.
9. Yedjou, C.G., et al., Basic apoptotic and necrotic cell death in human liver carcinoma (HepG(2) ) cells
induced by synthetic azamacrocycle. Environmental Toxicology, 2012. 10.1002/tox.21786: p. 1-7.