BCECF (solution)

Guide (The following is our recommended solution. This solution is merely a guideline and should be modified according to your specific needs.)
Prepare a 5-10 μM dye solution in the buffer (recommended is a serum-free HEPES buffer containing 0.1-0.2% BSA, with a pH of 7.4). (BCECF can be introduced into the cells by methods such as patch clamp and microinjection.)
2. Add 1000 μL per well (for 6-well plate), 100 μL per well (for 96-well plate), or 25 μL per well (for 384-well plate) of the BCECF staining solution to the cell-containing cell plate.
3. Place the dye loading plate in a cell culture box and incubate it in the dark for approximately 30 minutes (at 37°C and 5% CO?); after the incubation is complete, wash the cells twice with preheated PBS/buffer to remove the extracellular free dye, and then add preheated culture medium and continue incubation for 10-15 minutes to ensure that the intracellular AM groups are completely hydrolyzed.
4. By monitoring the fluorescence at Ex/Em = 490/535 nm (pH-sensitive) and 430/535 nm (pH-insensitive reference), the F???/F??? ratio is calculated to determine the pH; for each experiment, a standard curve needs to be established using a high-potassium buffer solution containing nigericin (pH gradient from 6.0 to 8.0) to achieve quantitative detection.

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