| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| 25mg |
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| Other Sizes |
| Targets |
No pharmacological target; BCECF is a fluorescent probe that binds to intracellular components non-specifically. Its fluorescence is quenched or enhanced by protonation/deprotonation of its phenolic groups. The ratio of emissions at 535 nm (upon excitation at 490 and 440 nm) is linearly related to pH over a wide range. It has been validated as a non-radioactive alternative to 51Cr-release cytotoxicity assays, where it measures membrane integrity by dye retention.
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| ln Vitro |
Advice (This is our suggested protocol, which should be adjusted to suit your particular circumstances as it simply offers guidance). 1. Prepare a stock solution of 2–20 mM BCECF in DMSO and store it at -20°C. 2. Make a buffer solution (5–50 μM BCECF dye loading) using Hepes (pH 7.4) or PBS. 3. To put the BCECF dye in the cell plate, add 1000 μL/well (6-well plate), 100 μL/well (96-well plate), or 25 μL/well (384-well plate). 4. The dye-loaded plate should be in the cell culture incubator for around half an hour. 5. Rinse and use buffer in place of dye loading solution. 6. For ratiometric measurements, watch fluorescence at Ex/Em = 490/535 nm or 430/535 nm to perform a pH determination.
In vitro, BCECF is used to calibrate pH measurements by preparing standard buffers of known pH (from pH 5.5 to 8.5) containing the dye, and measuring fluorescence ratios. The calibration curve is used to convert experimental ratios to pH values. Its fluorescence is insensitive to changes in ionic strength, temperature, and most common ions. The dye is photostable and has low cellular toxicity at typical loading concentrations (1–5 µM). |
| ln Vivo |
Not applicable as a therapeutic. BCECF is used only in research to monitor pHi changes in response to drugs, hormones, or metabolic alterations. It has been used in vivo to measure pH in tumors using intravital microscopy, but this is limited to preclinical imaging studies.
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| Enzyme Assay |
Cell-free assays: prepare a series of pH buffers (e.g., citrate, phosphate, Tris) from pH 5.0 to 8.5, containing 1 µM BCECF (free acid). Measure fluorescence at excitation 490 nm and 440 nm (emission 535 nm) using a fluorometer or plate reader. Plot the 490/440 ratio versus pH and fit a sigmoidal curve to obtain pKa. For cytotoxicity assays, BCECF-AM is not used cell-free.
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| Cell Assay |
Cells (e.g., endothelial cells, neurons) are incubated with 2–5 µM BCECF-AM in culture medium for 30–60 min at 37°C. After washing, cells are placed in a buffer (e.g., Krebs-Ringer) and fluorescence is measured on a plate reader with dual excitation filters. To calibrate, cells are treated with nigericin (10 µM) in high-K+ buffers of known pH, and ratios are recorded. Changes in pHi following addition of test compounds (e.g., ammonium chloride, weak acids) are monitored over time.
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| Animal Protocol |
In vivo, BCECF can be injected intravenously or applied topically in animal models for imaging of tissue pH. For example, BCECF-AM is infused into tumors via a microdialysis probe or instilled in the eye to measure corneal pH. Fluorescence is imaged using a microscope or endoscope. However, use is limited due to rapid clearance and tissue autofluorescence. No standardized animal protocols exist for therapeutic purposes.
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| ADME/Pharmacokinetics |
Molecular formula C27H20O11, MW 520.44. Appearance: solid. Solubility: DMSO (≥10 mg/mL), water (limited). BCECF-AM (acetoxymethyl ester) has MW 684.65 and is more soluble in DMSO. Store desiccated at -20°C, protected from light. Purity ≥90%. Excitation: 440/490 nm, Emission: 535 nm. The ester form is hydrolyzed in solution; prepare fresh aliquots.
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| Toxicity/Toxicokinetics |
No toxicity at working concentrations; BCECF-AM may have mild mitochondrial effects at high concentrations (>20 µM). The free acid is not membrane-permeant and is non-toxic. No genotoxicity or carcinogenicity reported. Standard safety precautions for handling fluorescent dyes apply.
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| References | |
| Additional Infomation |
BCECF is a research reagent, not a drug. It is the most widely used fluorescent pH probe and is available from multiple commercial suppliers. It has been a standard tool in cell physiology for decades. Its ratiometric capability makes it superior to single-wavelength indicators. It is also used in high-throughput screening for compounds affecting pH homeostasis.
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| Molecular Formula |
C27H20O11
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|---|---|
| Molecular Weight |
520.4411
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| Exact Mass |
520.101
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| CAS # |
85138-49-4
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| Appearance |
Yellow to orange solid powder
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| Density |
1.67g/cm3
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| Boiling Point |
936.2ºC at 760mmHg
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| Flash Point |
318.2ºC
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| Index of Refraction |
1.743
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| LogP |
3.398
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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: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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) |
DMSO : ~62.5 mg/mL (~120.09 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.00 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9215 mL | 9.6073 mL | 19.2145 mL | |
| 5 mM | 0.3843 mL | 1.9215 mL | 3.8429 mL | |
| 10 mM | 0.1921 mL | 0.9607 mL | 1.9215 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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