BCECF Acetoxymethyl ester

Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
The pH-sensitive fluorescent dyes to measure cytosolic pH^[2][3].
1.Prepare a 5 mM stock solution of BCECF Acetoxymethyl ester in DMSO.
2.Prepare a 3-5 μM BCECF Acetoxymethyl ester dye-loading solution in NMG buffer (10?mM HEPES, 60?mM KCl, 3?mM MgCl2, pH 6.8) or PBS.
3. Add BCECF Acetoxymethyl ester dye-loading solution (3-5 μM) into the cell plate for 30-60 min at 37°C in the dark.
4. Cells are washed three times with PBS.
5. Run the pH assay by monitoring the fluorescence at Ex/Em = 490/535 nm or 440/535 nm for ratio measurements.

Standard Curve Determination:
1. pH calibration buffer: 130?mM KCl, 1?mM MgCl2, 15?mM HEPES, 15?mM MES (HY-D0858), 10?μM Nigericin sodium salt (HY-100381), and 10?μM Valinomycin (HY-N6693) (to balance the intracellular and extracellular pH). Adjust to pH 6.5, 7.0, 7.5, 8.0, and 8.5 with NaOH or HCl.
2. Incubate the stained cells in calibration buffer at pH 6.5, 7.0, 7.5, 8.0, and 8.5 for 10 minutes, respectively.
3. Measure the F490/F440 ratio at each pH value. Plot a standard curve with pH as the horizontal axis and fluorescence ratio as the vertical axis (this curve should be sigmoidal and can be fitted using the Henderson-Hasselbalch equation).
4. Substitute the fluorescence ratio (R) of the unknown sample measured in the experiment into the standard curve to determine the corresponding pH value.

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