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BCECF-AM, also called 2',7'-Bis- (2-Carboxyethyl)-5- (and-6)-Carboxyfluorescein, Acetoxymethyl Ester, is a novel and potent fluorescent indicator which is cell membrane permeable and is thus widely used as a fluorescent marker/indicator for measurement of intracellular pH. It is the Acetoxymethyl Ester form of BCECF.
| Targets |
Fluorescent indicator/dye for intracellular pH.
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|---|---|
| ln Vitro |
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. |
| Enzyme Assay |
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. |
| Cell Assay |
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). |
| References |
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| Additional Infomation |
The fluorescent dye 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) has been widely used as an indicator of cytosolic pH. Here we report that BCECF localizes to hydrogenosomes (hydrogen-generating organelles found in several phylogenetically separate groups of anaerobic protists) in Trichomonas vaginalis, where it was observable by fluorescence microscopy. Its cellular location was confirmed by treatment of BCECF-loaded cells with diaminobenzidine and hydrogen peroxide together with UV illumination. This produced an osmiophilic precipitate in the matrix of hydrogenosomes, observable by electron microscopy. Use of a short (7.5 min) loading period, loading on ice, use of concentrations of BCECF (acetoxymethyl ester) down to 10 nM, and inclusion of the anion channel blockers probenicid or sulfinpyrazone, or the K+/H+ ionophore nigericin in the loading buffer all failed to prevent hydrogenosomal accumulation of BCECF. This uptake was best observed when intact cells were loaded with the ester form of BCECF, but could also be seen using free BCECF following either incubation with ruptured cells or electroporation of intact cells. Hydrogenosomal BCECF loading was also obtained with washed cell lysates, without cytoplasm or metabolic substrates. We tested a range of other fluorogenic dyes designed for cytosolic labeling, and found that the calcium indicator fura-2 (acetoxymethyl ester) and the cell viability marker fluorescein diacetate also labeled hydrogenosomes. The results illustrate a novel use for BCECF as a fluorescent marker for hydrogenosomes (the first such marker), but present a warning against the indiscriminate use of fluorogenic ester dyes to measure properties of the cytosol in hydrogenosome-containing organisms - the dyes may also be indicating the properties of the hydrogenosome.[1]
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| Molecular Formula |
C42H40O21
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|---|---|
| Molecular Weight |
880.754400000001
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| Exact Mass |
880.206
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| CAS # |
117464-70-7
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| PubChem CID |
53229972
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| Appearance |
Orange to red solid powder
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
939.2±65.0 °C at 760 mmHg
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| Flash Point |
368.9±34.3 °C
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| Vapour Pressure |
0.0±0.3 mmHg at 25°C
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| Index of Refraction |
1.602
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| LogP |
0.9
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
21
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| Rotatable Bond Count |
27
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| Heavy Atom Count |
63
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| Complexity |
1610
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
NTECHUXHORNEGZ-UHFFFAOYSA-N
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| InChi Code |
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
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| Chemical Name |
acetyloxymethyl 3',6'-bis(acetyloxymethoxy)-2',7'-bis[3-(acetyloxymethoxy)-3-oxopropyl]-3-oxospiro[2-benzofuran-1,9'-xanthene]-5-carboxylate
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| Synonyms |
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;
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.1354 mL | 5.6770 mL | 11.3540 mL | |
| 5 mM | 0.2271 mL | 1.1354 mL | 2.2708 mL | |
| 10 mM | 0.1135 mL | 0.5677 mL | 1.1354 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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