Fluorescent indicator/dye for intracellular pH.
BCECF-AM does not have a specific biological target as a therapeutic agent. It is a fluorescent probe designed for pH measurement in living cells. The acetoxymethyl ester (AM) group enables cell permeability by masking the negative charges of the carboxyl groups. Once inside the cell, esterases cleave the AM groups, converting BCECF-AM into the charged, membrane-impermeant BCECF, which remains trapped intracellularly. BCECF's fluorescence intensity varies with pH, allowing quantitative pH measurements.

Fully treated cells show hydrogenosomes with an electron-dense deposit which aggregates to a variable extent.The staining is observed in the interior of hydrogenosomes in some instances. It is also seen by microscopy that the K+/H+ ionophor nigericin does not inhibit hydrogenosomal loading with BCECF-AM.
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 (e.g. 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.
BCECF-AM is a non-fluorescent cell-permeable ester of BCECF that, on hydrolysis by cytosolic esterases, yields the intracellularly trapped pH-indicator BCECF. BCECF exhibits pH-dependent fluorescence, with excitation maxima at approximately 503 nm (pH-independent) and 439 nm (pH-dependent), and emission at 530 nm. The ratio of fluorescence at the two excitation wavelengths provides a ratiometric measurement of intracellular pH that is independent of dye concentration and cell thickness. The compound is widely used as a fluorescent marker/indicator for measurement of intracellular pH.

No in vivo activity data are documented for BCECF-AM as a therapeutic agent. The compound is a research tool used for intracellular pH measurements in isolated cells and tissues. It is not intended for therapeutic applications. In vivo applications of BCECF-AM are limited due to its rapid hydrolysis and the challenge of targeting specific tissues. The compound is primarily used in ex vivo and in vitro experimental settings.

Effect of ET-1on Na+/H+ exchange[2]
A standard ammonia pulse technique was used to measure NHE activity (Figure 2A). PASMCs loaded with BCECF were perfused at a rate of 1 mL/min with Solution 1 containing (in mmol/L): 130 NaCl, 5 KCl, 1MgCl2, 1.5 CaCl2, 10 glucose and 20 HEPES with pH adjusted to 7.4 with NaOH at 37°C. Baseline pHi was measured for 2 min before cells were briefly exposed to NH4Cl (ammonium pulse) by perfusing with Solution 2 containing (in mmol/L): 110 NaCl, 20 NH4Cl, 5 KCl, 1MgCl2, 1.5 CaCl2, 10 glucose, 20 HEPES at a pH of 7.4 using NaOH for 3 min. The ammonium pulse caused alkalinization due to influx of NH3 and buffering of intracellular H+ (Fig 2A). Washout of NH4Cl in the absence of extracellular Na+ using a Na+- and NH4 +- free solution containing (in mmol/L): 130 choline chloride, 5 KCl, 1MgCl2, 1.5 CaCl2, 10 glucose and 20 HEPES at a pH of 7.4 using KOH for 10 min results in acidification due to rapid diffusion and washout of NH3. The external solution was then switched back to Na+-containing Solution 1 for 10 min. Re-addition of extracellular Na+ allows activation of Na+/H+ exchange and recovery from acidification to basal levels. The rate of Na+-dependent recovery from intracellular acidification (change in pH over 2 min) corresponds to NHE activity.
In vitro enzyme assays for BCECF-AM are not typically performed as it is not an enzyme inhibitor. The compound is used as a substrate for esterases, which cleave the AM groups to release the fluorescent BCECF. For quality control and characterization, the hydrolysis of BCECF-AM by esterases can be monitored by fluorescence spectroscopy. The increase in fluorescence at 530 nm upon excitation at 503 nm indicates successful de-esterification. The compound is stable in dry form and should be protected from moisture and light.

Intracellular pH Measurements[2]
PASMCs were placed in a laminar flow cell chamber perfused with HBSS with pH adjusted to 7.4. pHi was measured in cells incubated with the membrane permeant (acetoxymethyl ester) form of the pH-sensitive fluorescent dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) for 60 min at 37°C under an atmosphere of 20% O2-5% CO2. Cells were 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 was 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 was 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 was returned through the objective and detected by an imaging camera. An electronic shutter was used to minimize photobleaching of dye. Protocols were executed and data collected on-line with InCyte software. pHi was estimated from in situ calibration after each experiment. Cells were perfused with a solution containing (in mmol/L): 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 was created from fluorescence measured as pHi was adjusted with KOH from 6.5 to 7.5. Intracellular H+ ion concentration ([H+]i) was determined from pHi using the formula: pHi  =  −log ([H+]i).
Cell-based assays with BCECF-AM are performed to measure intracellular pH. Cells are loaded with BCECF-AM by incubating them with the dye (typically 1-10 μM) in culture medium for 15-60 minutes at 37°C. The AM groups are cleaved by intracellular esterases, trapping BCECF inside the cells. After loading, cells are washed to remove extracellular dye. Fluorescence is measured using a fluorescence microscope, plate reader, or flow cytometer. Excitation at 440 nm and 490-505 nm with emission at 530 nm is used. The ratio of emission at the two excitation wavelengths is calculated and converted to pH using a calibration curve.

No animal models are documented for BCECF-AM as it is a fluorescent dye rather than a therapeutic agent. The compound is used for ex vivo measurements in isolated tissues or for in vivo imaging in certain research applications. For in vivo imaging, BCECF-AM may be injected locally or systemically, but its rapid hydrolysis and clearance limit its utility. The compound is primarily used in cell-based and tissue-based assays for pH measurement.

BCECF-AM has a molecular weight of approximately 880.75. It is a mixture of isomers with the molecular formula C80H72O38. The compound is light-sensitive and should be protected from light during handling and storage. It is soluble in DMSO and should be stored as a stock solution in DMSO at -20°C, protected from light and moisture. The compound is hygroscopic and should be handled under dry conditions. Solutions should be prepared fresh before use. The dye is membrane-permeable due to the AM ester groups.

No specific toxicity data are documented for BCECF-AM. The compound is a fluorescent dye used in cell biology research and is generally considered non-toxic at the concentrations used for pH measurements (1-10 μM). However, the AM groups may be hydrolyzed to release formaldehyde, which could be toxic at high concentrations. Standard laboratory safety practices should be followed when handling the compound. The compound is intended for research use only and is not approved for human therapeutic applications.

[1]. Analysis of the uptake of the fluorescent marker 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) by hydrogenosomes in Trichomonas vaginalis. Eur J Cell Biol. 1998 Jun;76(2):139-45.

[2]. Endothelin-1 Augments Na+/H+ Exchange Activity in Murine Pulmonary Arterial Smooth Muscle Cells via Rho Kinase. PLoS One. 2012; 7(9): e46303.

The fluorescent dye 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) has been widely used as an indicator of cytoplasmic pH. This article reports that BCECF is localized to the hydrogenosomes of Trichomonas vaginalis (hydrogen-producing organelles present in several phylogenetically different anaerobic protist groups) and can be observed by fluorescence microscopy. Treatment of BCEF-loaded cells with diaminobenzidine and hydrogen peroxide, combined with UV irradiation, further confirmed the cellular localization of BCEF. Following treatment, osmium-rich precipitates were generated in the hydrogenosome matrix, which could be observed by electron microscopy. Short loading times (7.5 min), loading on ice, using BCEF (acetoxymethyl ester) concentrations as low as 10 nM, and the addition of anion channel blockers such as probenecid or sulfinpyrazone, or the K+/H+ ion carrier nigrain to the loading buffer, failed to prevent the accumulation of BCEF in the hydrogenosomes. This uptake was most pronounced when intact cells were loaded with BCECF in ester form, but it was also observed when cells were loaded with free BCECF (whether incubated with ruptured cells or electroporated with intact cells). Loading of hydrogenosomes onto BCECF was also observed in washed cell lysates free of cytoplasm or metabolic substrates. We tested a range of other fluorescent dyes for cytoplasmic labeling and found that the calcium indicator fura-2 (acetoxymethyl ester) and the cell viability marker fluorescein diacetate could also label hydrogenosomes. The results suggest that BCECF can serve as a fluorescent marker for hydrogenosomes (the first such marker), but also serve as a warning against the indiscriminate use of fluorescent ester dyes to measure the properties of the cytoplasm of hydrogenosome-containing organisms—these dyes may also indicate the properties of hydrogenosomes. [1]
BCECF-AM is also known as 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester. It is a widely used fluorescent probe for measuring intracellular pH in living cells. The compound is cell-permeable and becomes trapped inside cells after hydrolysis by esterases. BCECF exhibits pH-dependent fluorescence, enabling ratiometric pH measurements. The compound is commonly used in studies of cellular physiology, apoptosis, drug transport, and pH regulation. It is intended for research use only.

C42H40O21

880.754400000001

880.206

117464-70-7

53229972

Orange to red solid powder

1.5±0.1 g/cm3

939.2±65.0 °C at 760 mmHg

368.9±34.3 °C

0.0±0.3 mmHg at 25°C

1.602

0.9

0

21

27

63

1610

0

CC(=O)OCOC1=CC2=C(C=C1CCC(=O)OCOC(=O)C)C3(C4=C(C=C(C=C4)C(=O)OCOC(=O)C)C(=O)O3)C5=C(O2)C=C(C(=C5)CCC(=O)OCOC(=O)C)OCOC(=O)C

NTECHUXHORNEGZ-UHFFFAOYSA-N

InChI=1S/C42H40O21/c1-22(43)52-17-57-34-15-36-32(13-27(34)7-10-38(48)59-19-54-24(3)45)42(31-9-6-29(12-30(31)41(51)63-42)40(50)61-21-56-26(5)47)33-14-28(8-11-39(49)60-20-55-25(4)46)35(16-37(33)62-36)58-18-53-23(2)44/h6,9,12-16H,7-8,10-11,17-21H2,1-5H3

acetyloxymethyl 3',6'-bis(acetyloxymethoxy)-2',7'-bis[3-(acetyloxymethoxy)-3-oxopropyl]-3-oxospiro[2-benzofuran-1,9'-xanthene]-5-carboxylate

BCECF-acetoxymethyl; MFCD00036969; acetyloxymethyl 3',6'-bis(acetyloxymethoxy)-2',7'-bis[3-(acetyloxymethoxy)-3-oxopropyl]-3-oxospiro[2-benzofuran-1,9'-xanthene]-5-carboxylate; Spiro[isobenzofuran-1(3H),9'-[9H]xanthene]-2',7'-dipropanoic acid, 3',6'-bis(acetyloxy)-5(or 6)-[[(acetyloxy)methoxy]carbonyl]-3-oxo-, 2',7'-bis[(acetyloxy)methyl] ester; ...; BCECF/AM - CAS 117464-70-7;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)