FluoForte exhibits significantly brighter fluorescence intensity over Fluo-4 AM and Fluo-3 AM. Hela cells were seeded overnight at 40,000 cells per 100µl per well in a 96-well black wall/clear bottom costar plate. The growth medium was removed, and the cells were incubated with 100µl of 4µM Fluo-3 AM, Fluo-4 AM or FluoForte™ AM in HHBS at 37°C, 5% CO2 incubator for 1 hour. The cells were washed twice with 200µl HHBS, ATP (20 µl/well) was added to achieve concentrations of 100nM with dye efflux inhibitor, then immediately imaged with a fluorescence microscope (Zeiss) using FITC channel.