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.
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