Many cell-based assays require the quantification of cell density in order to measure cytotoxicity or proliferation. Quantification can be accomplished directly by counting the cells or indirectly via colorimetric changes following substrate modification. In cases where there are few experimental conditions, direct cell counting can be carried out manually using a hemacytometer. With larger scale experiments, an automated cell counter (such as a Coulter Counter) is preferred. Indirect methods (i.e. colorimetric) of cell quantification usually involve the bioreduction of a tetrazolium salt by living cells to a colored formazan product.
I recently tried a cell proliferation assay that employs the tetrazolium salt/formazan method but without any of the complications of previously developed assays. Promega offers the CellTiter 96 AQueous One Solution Cell Proliferation Assay that uses a novel tetrazolium compound, MTS (abbreviations below), in combination with an electron coupling reagent, PES, to produce a colorimetric change. According to Promega, PES is chemically stable, allowing it to be combined with MTS to form a single-solution cell proliferation reagent. Cell proliferation assays offered in the past employed the tetrazolium compound MTT and an electron coupling reagent that was supplied separately. In addition, the formazan resulting from the bioreduction of MTT was insoluble in culture media, necessitating an additional solubilization step. With the CellTiter 96 One Solution assay, the formazan resulting from the bioreduction of MTS is soluble in media, eliminating the extra step. In fact, there are only two steps in the Promega assay: 1) add the One-Solution reagent to the wells of a 96-well tray containing cells and 2) measure the color change at 490 nm after 1-4 h of incubation.
I tested the CellTiter 96 proliferation assay on ADLC-5M2 lung adenocarcinoma cells. On Day 1, I plated in triplicate 500, 1000, 2000, 5000, 10000, 20000, 50000, or 100000 cells per well in 100 ul RPMI 1640/10%FBS in a 96-well plate. 
On day 2, I added 20 ul of the One-Solution Reagent to each well using a multichannel pipet, swirled the plate to mix, and returned it to the incubator. After 60 min, I measured the absorbance at 492 nm, which was the closest wavelength to the recommended 490 nm on our plate reader. As shown in the figure, the results were excellent. The assay appeared to have a linear response with 2000 - 20000 cells/well. However, since I only allowed the reaction to proceed for 60 min, it is likely that the lower limit would extend to fewer than 2000 cells/well with longer incubation times. Even at 60 min, the absorbance in the wells with 500 cells/well (0.174 0.004) was clearly different from that in the wells without cells (0.115 0.003).
In summary, Promega’s CellTiter 96 AQueous One Solution Cell Proliferation Assay is incredibly easy to use, quite sensitive and produces very clean data. In addition, depending on the size of the kit, the assay can be very economical, ranging in cost from around 14 cents/assay to 30 cents/assay. I highly recommend it.
Abbreviations
MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
PES: phenazine ethosulfate
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Michael Campa, Ph.D.
Assoc. Research Professor of Radiology
Duke University Medical Center