Promega’s CellTiter-Glo® Luminescent Cell Viability Assay determines the number of viable cells in a culture by quantification of ATP. There is a direct relationship between the number of viable cells and the amount of ATP present. This kit’s linear range is from 0 to 50,000 cells per well.
The experiment is quite simple. First, cells are seeded in a 96 well plate with 1 to 5,000 cells per well. Second, cells are treated with the experimental drug for 2 to 3 days. Third, the detection reagent is prepared by combining the CellTiter Glo® Buffer with CellTiter Glo® Substrate. Fourth, an equal volume of CellTiter-Glo® Reagent is added to each well of cell culture medium (you do not need to change medium). I used 100 ul in each well of a 96 well plate. After mixing for 2 minutes, the plate sits at room temperature for 10 minutes to stabilize the luminescence. Fifth, the luminescence is read with integration time set for 0.25 to 1 second. Since regents are added into the growth media directly, both adherent and suspended cells can be assayed with this kit. In my experiments, I use HUVEC cells (which are adherent cells) treated with VEGF analogs for comparison of the effects on cell proliferation.
You can use different concentrations of ATP or create standard curves using serial dilutions. In my experiments, I do not need absolute cell numbers; I am looking for relative changes. I use a 12 point serial dilution, with each point in triplicate, for drug treatment and then compare treated cells to untreated controls. Non-linear regressions are analyzed by GraphPAD Prism 4 software.
The luminescent signal generated by the CellTiter Glo® Assay is stable. The protocol claims a half-life more than 5 hours. I have found this to be true. I have read the plate at 10 minutes, 30 minutes and 90 minutes. The signal at 30 minutes is ok, 90 minutes is best. At about 10 minutes, it appears that the reaction is not finished yet as the signal variation is a little too big.
Tricky steps: Temperature is important in the reaction. Equilibrate all plates and reagents to room temperature. Two hours before performing the assay, take the reagents out of the freezer and put the buffer into a 37°C water bath to thaw. Wait for the buffer to reach room temperature before mixing it with the substrate. 30 minutes before performing the assay, take the cell culture plate out of the incubator, separate the plates in a sterile hood and let each well reach room temperature.
Xiang Yang
Research Scientist
Georgetown University
Lombardi Cancer Center