Available Methods of Assessing Viability
Researchers either detect an antigen which is present in proliferating cells but absent in
non-proliferating cell.
1.
2.
3.
4.
5.
Trypan Blue exclusion,
Neutral Red inclusion, and .
Crystal violet inclusion
Cell death is measured by the quantitation of 51Cr release.
alamarBlue is used to assess viability in response to toxic agents.
1. Trypan Blue exclusion, Neutral Red inclusion, and crystal violet inclusion.
Measuring percentage Trypan Blue exclusion can rapidly assess cell viability and confirm
whether cell culture conditions are optimal. Cells that are viable exclude Trypan Blue, while
cells that have died are stained by this dye. Trypan Blue exclusion measurement is also
useful for determining whether cells in culture have escaped breakage or disruption (Wright
et al., 1992) and to assess apoptosis (Wiley et al., 1995). Trypan Blue exclusion
measurement therefore has utility in assessing viability and also in assessing cell mortality
in response to environmental insult. The staining procedure with this dye is simple.
Determination of percentage Trypan Blue exclusion requires only a light microscope.
Typically, a small percentage of cells are harvested for Trypan Blue exclusion measurement.
Determination of Trypan Blue exclusion is performed at one or several predetermined
time points.
2. Neutral Red Staining
Neutral Red stains viable cells. This dye is absorbed by viable cells and is concentrated in
the lysosomes. Quantitation of Neutral Red staining therefore has utility in monitoring
cytotoxicity assays (Elliot and Auersperg, 1993; Modha et al., 1993). Results obtained when
measuring viability by Neutral Red uptake correlate well with viability measurements by
Trypan Blue exclusion (Modha etal., 1993).
As with determination of Trypan Blue exclusion, determination of percentage Neutral Red
staining requires only a light microscope. A small percentage of cells are harvested for
Neutral Red staining. Staining with Neutral Red is performed at one or several
predetermined time points.
3. Crystal Violet Inclusion
Crystal violet inclusion measurement detects cell lysis. This dye stains viable cells that
adhere to their culture vessel. Lysed cells simply fall away from their vessel surface and are
not stained by this dye.
Crystal violet is used in a standard bioassay for TNF-a. In this bioassay, fibroblasts are
grown as a monolayer in the presence of actinomycin D. To these cells, increasing
concentrations of TNF-a are added. Cells that are lysed in the presence of TNF-a are not
subsequently stained by crystal violet, whereas intact cells are stained by this dye. Crystal
violet-stained cells are then detected by spectrophotometric measurement (Coligan et al.,
1996). This method has the limitation of detecting only adherent cells and only permits
measurement at a single predetermined time point.
In other words, no continuous monitoring can be performed by this method.
4. Cell death is measured by the quantitation of 51Cr release.
Cell death is measured by the quantitation of chromium (51Cr) release from labeled target
cells.
In this assay, living cells nonspecifically incorporate and retain the radionuclide. As the cells
are killed, 51Cr is released into the culture medium and is quantitated by scintillation
counting.
Examples of use of the chromium release assay include the measurement of the cell killing
ability of cytotoxic T cells, ADCC, NK cell activity (Frey et al., 1991) and the measurement
of apoptosis (Wright et al., 1992).
The handling of 51Cr has several disadvantages which include the cost, equipment needs,
safety and environmental hazards associated with the handling of radioactive chromium.
5. alamarBlue is used to assess viability in response to toxic agents
The innate metabolic activity of cells can be monitored with alamarBlue. In the presence of
added toxic compounds such as antimicrobial agents, this innate metabolic activity ceases.
Procedures which cite the use of alamarBlue in assays of efficacy of antimicrobial agents
call for the determination of alamarBlue reduction at a single predetermined time point.
Examples of this use which include susceptibility testing of yeast and bacteria to
antimicrobial agents are discussed in detail below.
The reduction of alamarBlue appears to require uptake into the cell. To test whether cell
uptake is required, two cultures of A549 cells were grown to confluency in RPMI 1640
medium contained in T25 flasks. The spent media were removed from these cultures and
replaced with fresh media. Fresh media was also added to sterile flasks which contained no
cells. All flasks were then incubated at 37°C, 5% CO2 for four hours. After the four hour
incubation period, alamarBlue was added to all flasks. The control T flask containing no
cells was left in the upright position. One of the flasks containing cells was left in the
upright position so that cell culture medium supplemented with alamarBlue bathed the cells.
The other flask containing cells was turned upside down such that that cell culture medium
supplemented with alamarBlue no longer was in contact with the cells. Of the three flasks,
only the flask which contained cells bathed in medium supplemented with alamarBlue
reduced alamarBlue. This observation demonstrates that alamarBlue is not reduced by
compounds in the cell culture medium and implicates metabolism within the cells under
study as the site of alamarBlue reduction.
There are two ways to monitor alamarBlue reduction: by measuring absorbance
spectrophotometrically or by measuring fluorescence.
Because the absorption spectra of the oxidized (blue) and the reduced (red) forms of
alamarBlue overlap, the absorbance is measured at two wavelengths. These wavelengths
are 570 and 600 nm, where the reduced and oxidized forms absorb, respectively.
Alternatively, 540 and 630 nm may be used. In measuring alamarBlue reduction
spectrophotometrically, the plate reader is first blanked on a well containing medium only
at the two wavelengths. The absorbance of the medium without cells plus alamarBlue is
read at the two chosen wavelengths. Then the absorbance of the medium with cells
containing alamarBlue is read at the two chosen wavelengths.
In monitoring alamarBlue reduction spectrophotometrically, reduction is expressed as a
percentage (% Reduced).