Fluo-4 AM (solution)

Guide (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs)
. 1. Preparation of working solution
Dilute with buffer (e.g. HHBS containing 0.04% Poloxamer 407 (F127) (HY-D1005) ) (optimized according to the experiment). The corresponding stock solution can be diluted according to the actual situation. Note that if the solvent is DMSO, the cytotoxicity of DMSO must be considered, and a solvent control should be prepared; if the solvent is pure water, the working solution needs to be filtered and sterilized before adding cells.
Note: The working solution should be prepared and used immediately, and protected from light.

2. Cell staining

2.1 Count the cells and take 10⁶ cells from each sample (control and experiment/s).
2.2 Collect the cells (5 min, 3000×g, 4 °C) and wash once in PBS.
2.3 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.
2. 4 Wash the cells three times (2 min, 3000×g) with PBS and finally resuspend in 1 mL PBS.
2.5 Evaluate the staining by flow cytometry (Ex/Em = 485/526 nm).