BioTek July 2019 - 16

conditions for both the experimental and the calibration experiment must be the same for accurate
comparison. Alternatively the relative amount of absorbance generated under different conditions can
be compared within an experiment independent of
actually determining cell number. For example, as
observed in Figure 4, one could conclude that there
are significantly fewer viable cells with 24 hours exposure to 40 µM thiostrepton as compared to a 1 µM.

monochromators to provide wavelength specificity for both absorbance and fluorescence measurements. These hardware features allows the user to
measure the fluorescence or absorbance of samples
without the worry of having the necessary filters. In
addition spectral scans in either read mode can be
performed automatically by the microplate reader.
The reader is used in conjunction with Gen5™ Data
Analysis Software (BioTek Instruments). Besides controlling reader function, Gen5 collects and stores the
generated data. Gen5 is also capable of automatically plotting calibration curves, calculation unknown
concentrations, validating assay performance, and
providing assay reports.

There are a few precautions with using tetrazolium
slats for cell quantitation. Since the basis of the assay is the inherent dehydrogenase activity of viable
cells, treatments that affect dehydrogenase activity
may result in a discrepancy between the actual viable cell number and that determined using WST8. Tetrazolium salts such as WST-8 can also directly
interact with reducing agents such as DTT or b-ME;
resulting an increase in absorbance at 460 nm. If the
use of these agents cannot be avoided, background
subtraction of a media only (no cells) reaction can be
used to negate this. A slight spontaneous increase in
absorbance of media incubated with CCK-8 reagent
has been reported. The increase in background absorbance is dependant on the culture media, pH, incubation time, and exposure to light. Again, this can
be corrected for by subtraction of a no-cell blank.
Note that the CCK-8 reagent by itself does not under
go any increase in absorbance.

References
1.

Mosmann T (1983). Rapid colorimetric assay for cellular
growth and survival: application to proliferation and
cytotoxicity assays". Journal of Immunological Methods.
65: 55-63.

Kwok, J., S. Myatt, C. Marson, R.S. Coombes, D.
Constantinidou, E. Lam (2008) Thiostrepton Selectively
Targets Breast Cancer Cells through Inhibition of
Forkhead box M1 Expression. Molecular Cancer
Therapeutics. 7:2022-2032.

About the Author

The Synergy Multi-Mode Microplate Reader is
an ideal multipurpose reader. The reader uses

Paul Held, Ph.D., is Laboratory Manager, Applications
Dept., BioTek Instruments, Inc.

BioTek July 2019

Table of Contents for the Digital Edition of BioTek July 2019