BioTek July 2019 - 13

moiety into acid precipitable nucleic acid. While very effective
and specific, this method suffers
from a lengthy sample preparation procedure and from the use
of radioactivity, with its inherent
dangers, regulations and disposal costs. Several non-radioactive
assays have been developed.
These assays employ detection
technologies such as absorbance,
fluorescence, and luminescence.
Assays that detect the presence
of ATP, a labile high energy molecule only present in living cells
can be measured by the luminescence produce when it interacts with luciferase enzyme.
Substrates that are converted
directly from a colorless compound by cellular metabolism include the fluorescent compound
calcein, as well as many tetrazolium salts. Tetrazolium salts
are reduced enzymatically to
produce colored formazan dyes.
One of the first and most notable
of these salts is MTT, which produces a colored insoluble product that has to be solublized with
ethanol1. Improvements of the
tetrazolium substrates has produced compounds that have water soluble products; the first of
which is WST-1. This compound
has a maximal absorbance
wavelength of 438 nm. Further

improvements in the tetrazolium
salts have improved both reactivity and molar absorbtivity, along
with decreasing toxicity.
The Cell Counting Kit from
Dojindo uses WST-8 in conjunction with an electron mediator,
1-methoxy PMS, to assess viable
cells. The tetrazolium salt, WST8, is reduced through a reaction
with the reduced form of 1-methoxy PMS. This mediator resides
at the cell membrane and reacts
directly with NADH (nicotineamido adenine dinucleotide reduced
form) or NADPH (nicotineamido
adenine dinucleotide phosphate
reduced form) (Figure 1). NADH
and NADPH are generated from

Figure 1. WST-8 Assay Mechanism.

NAD+ or NADP+ by the reaction
of dehydrogenase enzymes and
their substrates, such as lactate
dehydrogenase and lactic acid
respectively. Therefore, the tetrazolium salt is utilized for the determination of the dehydrogenase activity or a substrate of the
dehydrogenase.
The solution of a WST-8 tetrazolium salt is almost colorless while
its formazan product gives a
strongly colored solution with a
peak absorbance at 460 nm.

Materials and Methods
A Cell Counting Kit (CCK-8) was
obtained from Dojino Molecular

BioTek July 2019

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