The CellTiter-Blue™ Reagent is a buffered solution containing highly purified resazuin. Resazuin is dark blue in color and has little intrinsic fluorescence. Metabolically active cells can reduce resazuin to resorufin, which is pink and highly fluorescent, with an exitation maximum of 579nm and an emission maximum of 584nm.
The experiment is simple. Begin by seeding cells in plates, culturing the cells and then treating them with the desired drug. After 2 to 3 days, add 20ul CellTiter-Blue™ Reagent per 100ul of culture medium to each well, shake briefly, incubate under standard cell culture conditions for 1 to 4 hours. Read the results in a fluorescence reader with an excitation of 530 to 570nm and an emission of 580 to 620nm. Results also can be read in an absorbance reader at 570nm and 600nm. The reaction can be stopped and stabilized with SDS. The plate then can then be stored at ambient temperature for up to 24 hours before reading. Because different cells have different metabolic ratios, you may find it optimal to measure fluorescence at different times.
The CellTiter-Blue™ Viability Assay is used to estimate the number of viable cells present in multiwell plates. It is suitable for both adherent and suspension cells. In my experiments, I am checking HUVAC cell proliferation after stimulation with VEGF analogs. This kit’s fluorescent signal linear range and lower limit of detection are dependent on the cells’ ability to reduce reazurin. It may be that HUVAC cells are not good at reducing reazurin. My experimental results show bigger variations than I have seen with other assays. Fluorescent and absorbance readings both show the same variations. Another possibility is that the cells did not lyse well, which also could affect the fluorescent and absorbance readings.
In my experiments, I did not need absolute cell numbers; I was looking only for relative changes. I used a 12 point serial dilution of my drugs for stimulation, each point was done in triplicate and the stimulated samples were compared to untreated controls. Non-linear regressions were analyzed by GraphPad Prism 4 statistical software.
Tricky steps: During the 1-4 hour reaction and after the reaction is stopped with SDS, the plate should be protected from light. I wrapped the plate in aluminum foil.
Xiang Yang
Research Scientist
Georgetown University
Lombardi Cancer Center