BCECF-Acetoxyemnethy ester 

BCECF-Acetoxyemnethy ester is a cell membrane permeable compound widely-used as a fluorescent indicator for intracellular pH. BCECF-Acetoxyemnethy ester 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^[1].

The pH-sensitive fluorescent dyes to measure cytosolic pH^[2][3].
1.Prepare a 5 mM stock solution of BCECF-Acetoxyemnethy ester in DMSO.
2.Prepare a 3-5 μM BCECF-Acetoxyemnethy ester dye-loading solution in NMG buffer (10 mM HEPES, 60 mM KCl, 3 mM MgCl2, pH 6.8) or PBS.
3. Add BCECF-Acetoxyemnethy 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.

880.76

C₄₂H₄₀O₂₁

Oil

Brown to reddish brown

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Room temperature in continental US; may vary elsewhere.

-20°C, protect from light

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

• [1]. Ozkan P, et al. A rapid method for measuring intracellular pH using BCECF-AM. Biochim Biophys Acta. 2002 Aug 15;1572(1):143-8.  [Content Brief]

  [2]. Chen F, et al. CYP51A1 drives resistance to pH-dependent cell death in pancreatic cancer. Nat Commun. 2025 Mar 7;16(1):2278.  [Content Brief]

  [3]. Pang X, et al. Weak acids produced during anaerobic respiration suppress both photosynthesis and aerobic respiration. Nat Commun. 2023 Jul 14;14(1):4207.  [Content Brief]