BCECF-AM

BCECF-AM is a cell membrane permeable compound widely used as a fluorescent indicator for intracellular pH. BCECF-AM could diffuse through the cell membrane and intracellular esterase cleave the ester bond releasing BCECF (HY-101882). BCECF allows measurements in the physiological pH range 6.0-8.0. Excitation ratio: 490/440 nm; Emission intensity: 535 nm. In Vitro:The pH-sensitive fluorescent dyes to measure cytosolic pH^[3][4].
1.Prepare a 5 mM stock solution of BCECF-AM in DMSO.
2.Prepare a 3-5 μM BCECF-AM dye-loading solution in NMG buffer (10 mM HEPES, 60 mM KCl, 3 mM MgCl2, pH 6.8) or PBS.
3. Add BCECF-AM 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.