( then analyzed using the GEMINI XS. ...)LIVE/DEAD Viability/Cytotoxicity Assay for Animal Cells Using the SpectraMax Gemini XS Fluorescence Microplate Reader (MaxLine Application Note #43)

RESULTS
Excitation and Emission Wavelength Optimization
The parts of the plates containing 2.5 x 10⁴ live and dead cells/well were scanned on spectrum mode to determine optimal Ex and Em wavelengths for the cal AM dye, as shown in Figure 1A. Next, this part of the plate was scanned to optimize wavelengths for the EthD-1 dye (Fig. 1B). The combination of Ex/Em wavelengths that showed the greatest difference between live and dead cells was chosen for future experiments. These include Ex485nm/Em525nm using a 515nm cutoff filter for cal AM stained live cells, and Ex525nm/Em620nm using a 590nm cutoff filter for EthD-1 stained dead cells. These settings were used for characterizing the performance of the assay.

Assay Performance
Plates were read using the well scan feature, taking readings at ten different positions in each well. The signal to background ratios were calculated by dividing the RFU value obtained for 2.5 x 10⁴cells by the value for the negative control (PBS only). The values for cal AM and EthD-1 were 6.2 and 8.1, respectively. All of the wells containing live cells, dead cells and PBS only were scanned under optimal Ex/Em conditions for each dye to determine the linearity (Tables 1A, B). The CV% for all cell dilutions was well below 10%. The linear curve for live cells was compared to that obtained with the same number of dead cells, both were scanned at 485/525 (optimal for cal AM) (Fig. 2A). In this scan, the background signal for dead cells remained constant for up to the maximum, 2.5 x 10⁴ cells/well. In addition, live and dead cell fluorescence was compared at 525/620 (Fig. 2B). In contrast, there was a small increase in signal from 103 to 2.5 x 10⁴ live cells/well. In other words, 2.5 x 10⁴ live cells/well produce a
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