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.