Factors to
Consider When Choosing a Cell Viability Assay:
Among
the many factors to consider when choosing a cell-based assay, the primary
concern for many researchers is the ease of use. Homogeneous assays do not
require removal of culture medium, cell washes or centrifugation steps. When
choosing an assay, the time required for reagent preparation and the total
length of time necessary to develop a signal from the assay chemistry should be
considered. The stability of the absorbance, fluorescence or luminescence
signal is another important factor that provides convenience and flexibility in
recording data and minimizes differences when processing large batches of
plates. Another factor to consider when selecting an assay is sensitivity of
detection. Detection sensitivity will vary with cell type if you choose to
measure a metabolic marker, such as ATP level or MTS tetrazolium reduction. The
signal-to-background ratios of some assays may be improved by increasing
incubation time. The sensitivity not only depends upon the parameter being
measured but also on other parameters of the model system such as the plate
format and number of cells used per well. Cytotoxicity assays that are designed
to detect a change in viability in a population of 10,000 cells may not require
the most sensitive assay technology. For example, a tetrazolium assay should
easily detect the difference between 10,000 and 8,000 viable cells. On the
other hand, assay model systems that use low cell numbers in a high-density
multiwell plate format may require maximum sensitivity of detection such as
that achieved with the luminescent ATP assaytechnology.For researchers using
automated screening systems, the reagent stability and compatibility with
robotic components is often a concern. The assay reagents must be stable at
ambient temperature for an adequate period of time to complete dispensing into
several plates. In addition, the signal generated by the assay should also be
stable for extended periods of time to allow flexibility for recording data.
For example, the luminescent signal from the ATP assay has a half-life of about
5 hours, providing adequate flexibility. With other formats such as the MTS
tetrazolium assay or the LDH release assay, the signal can be stabilized by the
addition of a detergent-containing stop solution. In some cases the choice of
assay may be dictated by the availability of instrumentation to detect
absorbance, fluorescence or luminescence. The Promega portfolio of products
contains an optional detection format for each of the three major classes of
cell-based assays (viability, cytotoxicity or apoptosis). In addition, results
from some assays such as the ATP assay can be recorded with more than one type
of instrument (luminometer, fluorometer or CCD camera).
Cost
is an important consideration for every researcher; however, many factors that
influence the total cost of running an assayare often overlooked. All of the
assays described above are homogeneous and as such are more efficient than
multistep assays. For example, even though the reagent cost of an ATP assay may
be higher than other assays, the speed (time savings), sensitivity (cell sample
savings) and accuracy may outweigh the initial cost. Assays with good detection
sensitivity that are easier to scale down to 384 or 1536well formats may result
in savings of cell culture reagents and enable testing of very small quantities
of expensive or rare test compounds. The ability to gather more than one set of
data from the same sample (i.e., multiplexing) also may contribute to saving
time and effort. Multiplexing more than one assay in the same culture well can
provide internalcontrols and eliminate the need to repeat work. For instance,
the LDH-release assay is an example of an assay that can be multiplexed. The
LDH-release assay offers the opportunity to gather cytotoxicity data from small
aliquots of culture supernatant that can be removed to a separate assay plate,
thus leaving the original assay plate available for any other assay such as gene
reporter analysis, image analysis, etc. Several of our homogeneous apoptosis
and viability assays can be multiplexed without transferring media, allowing
researchers to assay multiple parameters in the same sample well.
Reproducibility
of data is an important consideration when choosing a commercial assay.
However, for most cell-based assays, the variation among replicate samples is
more likely to be caused by the cells rather than the assay chemistry.
Variations during plating of cells can be magnified by using cells lines that
tend to form clumps rather than a suspension of individual cells. Extended
incubation periods and edge effects in plates may also lead to decreased
reproducibility among replicates and less desirable Z’-factor values.
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