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AAT Bioquest

BCECF, AM *UltraPure grade*

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Fax1-800-609-2943
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ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight808.69
SolventDMSO
Spectral properties
Extinction coefficient (cm -1 M -1)90000
Excitation (nm)504
Emission (nm)527
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200
Alternative formats
BCECF, AM

OverviewpdfSDSpdfProtocol


CAS
117464-70-7
Molecular weight
808.69
Extinction coefficient (cm -1 M -1)
90000
Excitation (nm)
504
Emission (nm)
527
Intracellular pH plays an important modulating role in many cellular events, including cell growth, calcium regulation, enzymatic activity, receptor-mediated signal transduction, ion transport, endocytosis, chemotaxis, cell adhesion and other cellular processes. pH-sensitive fluorescent dyes have been widely applied to monitor changes in intracellular pH in recent years. Imaging techniques that use fluorescent pH indicators also allow researchers to investigate these processes with much greater spatial resolution and sampling density that can be achieved using other technologies such as microelectrode. Among them, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) is the most popular pH probe since it can be used to monitor cellular pH ratiometrically. BCECF AM is the cell-permeable version of BCECF.

Platform


Fluorescence microscope

ExcitationFITC filter set
EmissionFITC filter set
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation490, 430
Emission535
Cutoff515
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode

Example protocol


PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

BCECF AM *UltraPure Grade* Stock Solution
Prepare a 2 to 20 mM stock solution of BCECF AM in high-quality, anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

BCECF AM *UltraPure Grade* Working Solution
On the day of the experiment, either dissolve BCECF AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a BCECF AM working solution of 5 to 50 µM in a buffer of your choice (e.g., Hanks and Hepes buffer).
Note     The nonionic detergent Pluronic® F-127 can be used to increase the aqueous solubility of AM esters. In the staining buffer, the final Pluronic® F-127 concentration should be approximately 0.02%. A variety of Pluronic® F-127 products can be purchased from AAT Bioquest. Avoid long-term storage of AM esters in the presence of Pluronic® F-127.
Note     If your cells contain organic anion-transporters, probenecid (1-2 mM) may be added to the dye working solution (final in well concentration will be 0.5-1 mM) to reduce leakage of the de-esterified indicators. A variety of ReadiUse™ probenecid products, including water-soluble, sodium salt, and stabilized solution, can be purchased from AAT Bioquest.

SAMPLE EXPERIMENTAL PROTOCOL

The following is a recommended protocol for loading BCECF AM into live mammalian cells. This protocol only provides a guideline, should be modified according to your specific needs.
  1. Prepare viable cells as desired.
  2. On the next day, add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of the BCECF AM working solution into the cell plate.
    Note     If your compound(s) interfere with the serum, replace the growth medium with fresh HHBS buffer (100 μL/well for 96-well plate or 25 μL/well for 384-well plate) before dye-loading.
  3. Incubate the dye-loaded plate in a cell incubator at 37 °C for 30 to 60 minutes.
  4. Replace the dye working solution with HHBS or buffer of your choice to remove any excess probes.
  5. Prepare the compound plates using HHBS or a buffer of your choice.
  6. Run the pH assay as desired and simultaneously measure fluorescence using either a fluorescence microscope equipped with a FITC filter set or a fluorescence plate reader at Ex/Em = 490/535 nm cutoff 515 nm. For ratio measurements, monitor fluorescence at Ex/Em1 = 430/535 nm cutoff 515 nm and Ex/Em2 = 505/535 nm cutoff 515 nm.
    Note     The compound addition is 50 μL/well (96-well plate) or 25 μL/well (384-well plate).
    Note     Assays should be completed within 3 to 5 minutes after compound addition. However, a minimum of 8 minutes is recommended for data collection.  

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of BCECF, AM *UltraPure grade* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM123.657 µL618.284 µL1.237 mL6.183 mL12.366 mL
5 mM24.731 µL123.657 µL247.314 µL1.237 mL2.473 mL
10 mM12.366 µL61.828 µL123.657 µL618.284 µL1.237 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


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spectrum

Spectral properties

Extinction coefficient (cm -1 M -1)90000
Excitation (nm)504
Emission (nm)527

Images


Citations


View all 2 citations: Citation Explorer
The F0F1 ATP synthase regulates human neutrophil migration through cytoplasmic proton extrusion coupled with ATP generation
Authors: Gao, Jun and Zhang, Tian and Kang, Zhanfang and Ting, Weijen and Xu, Lingqing and Yin, Dazhong
Journal: Molecular Immunology (2017): 219--226
Oxidative Stress-Activated NHE1 Is Involved in High Glucose-Induced Apoptosis in Renal Tubular Epithelial Cells
Authors: Wu, Yiqing and Zhang, Min and Liu, Rui and Zhao, Chunjie
Journal: Yonsei Medical Journal (2016): 1252--1259

References


View all 34 references: Citation Explorer
Simultaneous measurement of water volume and pH in single cells using BCECF and fluorescence imaging microscopy
Authors: Alvarez-Leefmans FJ, Herrera-Perez JJ, Marquez MS, Blanco VM.
Journal: Biophys J (2006): 608
Photophysics of the fluorescent pH indicator BCECF
Authors: Boens N, Qin W, Basaric N, Orte A, Talavera EM, Alvarez-Pez JM.
Journal: J Phys Chem A Mol Spectrosc Kinet Environ Gen Theory (2006): 9334
Drug efflux transport properties of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (bcecf-am) and its fluorescent free acid, bcecf
Authors: Bachmeier CJ, Trickler WJ, Miller DW.
Journal: J Pharm Sci (2004): 932
A rapid method for measuring intracellular pH using BCECF-AM
Authors: Ozkan P, Mutharasan R.
Journal: Biochim Biophys Acta (2002): 143
Detection of MRP functional activity: calcein AM but not BCECF AM as a Multidrug Resistance-related Protein (MRP1) substrate
Authors: Olson DP, Taylor BJ, Ivy SP.
Journal: Cytometry (2001): 105
In vivo application of intestinal ph measurement using 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (bcecf) fluorescence imaging
Authors: Marechal X, Mordon S, Devoisselle JM, Begu S, Guery B, Neviere R, Buys B, Dhelin G, Lesage JC, Mathieu D, Chopin C.
Journal: Photochem Photobiol (1999): 813
Fluorescence probe (BCECF) loading efficiency in human platelets depends on cell concentration: application to pHi measurements
Authors: Ruiz-Palomo F, Garcia C, Gomez M, Revenga M.
Journal: Clin Biochem (1999): 391
Analysis of the uptake of the fluorescent marker 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (bcecf) by hydrogenosomes in trichomonas vaginalis
Authors: Scott DA, Docampo R, Benchimol M.
Journal: Eur J Cell Biol (1998): 139
Simultaneous detection of cell volume and intracellular pH in isolated rat duodenal cells by confocal microscopy and BCECF
Authors: Weinlich M, Heydasch U, Mooren F, Starlinger M.
Journal: Res Exp Med (Berl) (1998): 73
BCECF in single cultured cells: inhomogeneous distribution but homogeneous response
Authors: Weinlich M, Theiss C, Lin CT, Kinne RK.
Journal: J Exp Biol (1998): 57