In Vitro: Fluo-4 AM is a fluorescent dye (λex=494 nm, λem=516 nm). Preloaded with Fuo-4 AM, a very bright fluorescence image is observed. In a parallel experiment with fluo-3 AM-loaded cells, the resulting fluorescence image, although clearly discernable in this case, is less bright.?Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs). ?1.?Count the cells and take 106 cells from each sample (control and experiment/s). ?2.?Collect the cells (5 min, 3000×g, 4 °C) and wash once in PBS. ?3.?Resuspend the cells in 0.5-ml PBS and add 0.5 μl of Fluo-4-AM (1 mM stock) to a final concentration of 1 μM. Incubate at 37 °C for 1 h. ?4.?Wash the cells three times (2 min, 3000×g) with PBS and finally resuspend in 1-ml PBS. Separate into 2 flow cytometry tubes—0.5 ml in each. ?5.?Evaluate the staining by flow cytometry and analyze the data by a software such as CellQuest software.