Comparison of microplate reader high-throughput cell viability assays using fluorescent, luminescent and absorbance detection

High-throughput cell viability assays are broadly used in screening experiments, e. g. for compound toxicity screens or to identify compounds that selectively kill cancer cells. Another common use is screening for environmental toxins. There are numerous assays available for probing cellular viability. Most of the assays are designed for workflows using microplates and optical detection methods like luminescence, fluorescence intensity or absorbance assays in microplate readers.

Here, we compared three different assays covering the 3 detection modes, namely CellTiter Glo 2.0 ® (Promega), Alamar blue ^TM and Vybrant ® MTT (both Thermo Fisher Scientific). The assays’ performance regarding dynamic range, lower limit of detection and upper limit of detection as well as handling were compared by means of a HeLa cell standard curve from 25-50,000 cells in 96- vs. 384-well plates.

Because of its add and read procedure, CellTiter Glo provides a seamless integration into automated workflows. Alamar Blue and MTT both require an incubation period of several hours, the addition of a stop solution and additional incubation time and mixing steps.

Although luminescence based CellTiter Glo showed the best lower limit of detection, all assays were affected by saturation on the upper limit of detection. This can probably be attributed to a limited substrate/cells ratio which indicates the need to adjust the assay procedure for higher cell numbers.