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BCECF-AM is a cell membrane permeable compound widely used as a fluorescent indicator for intracellular pH.

For research use only. We do not sell to patients.

BCECF-AM Chemical Structure

BCECF-AM Chemical Structure

CAS No. : 117464-70-7

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Based on 3 publication(s) in Google Scholar

Top Publications Citing Use of Products
  • Biological Activity

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BCECF-AM is a cell membrane permeable compound widely used as a fluorescent indicator for intracellular pH.

In Vitro

Fully treated cells show hydrogenosomes with an electron-dense deposit which aggregates to a variable extent.The staining is seen in the interior of hydrogenosomes in some instances. It is also observed by microscopy that the K+/H+ ionophor nigericin does not inhibit hydrogenosomal loading with BCECF-AM[1].
The pH-sensitive fluorescent dyes to measure cytosolic pH.
1.Prepare a 2 to 20 mM stock solution of BCECF-AM in DMSO.
2.Prepare a 5-50 μM BCECF-AM dye-loading solution in buffer solutions (HHBS or PBS).
3. Add 1000 μL/well (6-well plate),100 μL/well (96-well plate) or 25 μL/well (384-well plate) BCECF-AM dye-loading solution into the cell plate.
4. Incubate the dye-loading plate in a cell incubator for 30-60 minutes.
5. Wash and replace the dye-loading solution with buffers.
6. Run the pH assay by monitoring the fluorescence at Ex/Em = 490/535 nm or 430/535 nm for ratio measurements.

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

Molecular Weight







Orange to red

Emission (Em)


Excitation (Ex)





Room temperature in continental US; may vary elsewhere.


-20°C, protect from light

*The compound is unstable in solutions, freshly prepared is recommended.

Purity & Documentation

Purity: ≥99.0%

Cell Assay

PASMCs are placed in a laminar flow cell chamber perfused with HBSS with pH adjusted to 7.4. pHi is measured in cells incubated with the membrane permeant (acetoxymethyl ester) form of the pH-sensitive fluorescent dye BCECF-AM for 60 min at 37°C under an atmosphere of 20% O2-5% CO2. Cells are then washed with HBSS for 15 min at 37°C to remove extracellular dye and allow complete de-esterification of cytosolic dye. Ratiometric measurement of BCECF fluorescence is performed on a workstation consisting of a Nikon TSE 100 Ellipse inverted microscope with epi-fluorescence attachments. The light beam from a xenon arc lamp is filtered by interference filters at 490 and 440 nm, and focused onto the PASMCS under examination via a 20× fluorescence objective. Light emitted from the cell at 530 nm is returned through the objective and detected by an imaging camera. An electronic shutter is used to minimize photobleaching of dye. Protocols are executed and data collected on-line with InCyte software. pHi is estimated from in situcalibration after each experiment. Cells are perfused with a solution containing (in mM): 105 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, 20 HEPES-Tris and 0.01 nigericin to allow pHi to equilibrate to external pH. A two point calibration is created from fluorescence measured as pHi is adjusted with KOH from 6.5 to 7.5. Intracellular H+ ion concentration ([H+]i) is determined from pHi using the formula: pHi = −log ([H+]i).

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

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BCECF-AM Related Classifications

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    Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.

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