Fluo-4 AM

Fluo-4 AM is a cell-permeable Ca²⁺ (calcium ion) indicator (Ex/Em = 485/526 nm)^[1]. In Vitro:Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs)
. 1. Solution preparation^[1]
1.1 Preparation of storage solution
Solvent: DMSO
Concentration: 1 mM is recommended (e.g. weigh 100 μg Fluo-4 AM and dissolve in 88 μL solvent).
Storage: Use as soon as possible and avoid long-term storage.
1.2 Preparation of working solution
Dilute with buffer (e.g. HHBS containing 0.04% Poloxamer 407 (F127) (HY-D1005)) to 1 M (optimized according to the experiment).
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).