When it comes to performing cellular proliferation assays, choosing an assay type and readout method can make the difference between actually finding compounds or proteins of interest and not finding them. Sensitivity, price, and the speed of the assay need to be considered for efficient results. Avoiding or minimizing false positives and false negatives is also a high priority in choosing an assay. With all of this in consideration, our laboratory has chosen the Bright-Glo™ Luciferase Assay from Promega for our cellular proliferation assays.
The Bright-Glo™ Assay is based on the utilization of endogenous cellular luciferase levels to quantify the amount of cells. The supplied reagent both lyses the cells to release the luciferin and supplies Adenosine Tri-phosphate (ATP) to catalyze a phosphate transfer. The Luciferin-dependant transfer of the phosphate from ATP releases an energy emission which may be monitored on most any luminescent reader. It is important to note that this assay requires the transfection of cell lines to stably express the Luciferin protein. A disadvantage of transient transfection is a lack of transfection efficiency data; therefore, we recommend performing stable transfections on cell lines. Once stable cell lines are established, the assay results have shown to be highly reproducible. We have tested the reagent on over 100 cell lines including both adherent and suspension cell lines and have found this kit to yield useful results on both types. Signal intensity does vary from cell line to cell line; however, the signal to noise is most often large enough, >10 fold, to minimize significant noise fluctuations. In addition, we have not seen signal disruption due to the use of various media.
Upon considering how fast an assay can be run, this kit has the same speedy advantages that the Promega CellTiter-Glo™ Luminescent Cell Viability Assay (also reviewed in Biocompare) gives, but with one compromise: The half-life of the luminescent signal. Whereas with the CellTiter-Glo™ Assay, the signal half-life is 1 hour, the Bright-Glo™ assay signal has a half-life of only 30 minutes. In our hands, we've optimized the assay by reading plates 2 to 15 minutes after the addition of reagent. Although this may not make a difference for experiments containing a few plates, once 12 or more plates are used, this relatively short-lived signal requires that specific timing of reagent addition and plate reading be used considered an important variable for accurate readings.
In summation, the Bright-Glo™ Assay is a valuable resource for all types of laboratories, including both academic and industry focused institutions. It is a simple and easy assay to run, produces highly reproducible results, and does not require any expensive purchases for specific readers. The fact that it can be used with many cell types makes it an attractive assay, especially for experiments such as small molecule specificity screening across various cell types.