Fluorescence and luminescence cell viability assays. (A-C) Quantitation of cell viability of U937 cells after a 72-hour treatment with increasing concentrations of the antimitotic agent MMAE. (A) The cells were then lysed and incubated with CellTiter-Glo to measure the amount of ATP in the cells. This is a correlative readout for cell viability and proliferation. The plate was read on a microplate luminometer after we placed a custom-made plastic grid over the plate to prevent well-to-well light contamination. (B) U937 cells were labeled with CMFDA 24 hours before the addition of MMAE. After the 72-hour MMAE treatment, the plate was washed to remove dead cells and cellular debris and then scanned on an Image Xpress Velos laser scanner and the number of cells remaining was calculated. (C) U937 cells were dual stained with EB and AO to measure live, apoptotic, and necrotic cells simultaneously. This dual stain causes live cells to fluoresce green while apoptotic cells retain a red-orange fluorescence. (D) Visualization of the images used to acquire data for (C). Scale bar = 70 μm. (E) Quantitation of the effect of washing the plates after MMAE treatment and EB/AO staining on the number of dead versus live cells remaining on the plate surface reveals that the washing steps do not affect the overall counts for live or dead cells.