[CAS NO. 273221-67-3]  Fluo-4 AM

Fluo-4 AM is a fluorescent dye (λ _ex =494 nm, λ _em =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 ^[1] . 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.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.