BioTek July 2019 - 6

For example, BioTek recently demonstrated the cytotoxic effect of thiostrepton on mesothelial cells using
Invitrogen's Vybrant® MTT Cell Proliferation Assay Kit
and BioTek's Epoch™ Microplate Spectrophotometer.

enter cells but become reduced extracellularly by
electron transport at the cell membrane to produce
soluble formazan. For example, in WST-1 assays, the
WST-1 tetrazolium salt is reduced to a soluble form
of formazan in a reaction that is linked to the glycolytic production of NAD(P)H and is, therefore, directly related to metabolic activity of the cells.

Assaying ATP concentration
Proliferation can also be assayed by taking advantage of the tight regulation of the intracellular
concentration of ATP. Healthy cells synthesize ATP,
whereas unhealthy or dead cells contain little or no
ATP. The firefly enzyme luciferase uses ATP to oxidize
the substrate D-luciferin, resulting in light production. This bioluminescent system makes for a sensitive assay of the detection of ATP in cell cultures.
The assay usually begins with lysing the cells to release the intracellular stores of ATP, while also inhibiting ATPases to prevent the degradation of the released ATP. The amount of ATP is then measured by
adding a combination of luciferase and luciferin to
the lysed cell sample. As the enzyme luciferase uses
the available ATP to oxidize the substrate, it produces light proportional to the ATP concentration, allowing a sensitive measurement of healthy cells in a
cell sample using microplate readers.

Figure 2. The Epoch Microplate Spectrophotometer measures absorbance
in a range of 200 to 999 nm, enabling a diverse range of research and
analytical applications.

Other tetrazolium salts such as XTT and MTS are similar to MTT, but their reduced product is soluble in
culture media, eliminating the need for the extra solubilization step. For example, in XTT assays, healthy
cells convert XTT to a water-soluble, orange-hued
product of formazan in a complex bioreduction reaction. Alamar Blue redox dye is a type of resazurin
that enters live, viable cells and then becomes reduced intracellularly. The subsequent cell signals
can be detected using microplate readers.

Assaying DNA synthesis
Some workflows may benefit from readouts of DNA
synthesis as indicators of proliferation. Proliferating
cells are characterized in part by increased DNA synthesis activity. Traditionally, cells were incubated
with radioactive [3H]-thymidine, which was incorporated into DNA. However, today, nonradioactive

A similar reagent, known as water soluble tetrazolium salts (WSTs), are tetrazolium dyes that do not

https://www.biotek.com/assets/tech_resources/Quantification%20of%20Cell%20Viability%20App%20Note.pdf

BioTek July 2019

Table of Contents for the Digital Edition of BioTek July 2019