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AAT Bioquest

RatioWorks™ BCFL, AM *Superior replacement for BCECF*

Standard curve created using BCFL, AM (Cat# 21190) and BCECF,AM (Cat#21202) with Intracellular pH Calibration Buffer Kit. Hela cells were incubated with 5 uM BCFL, AM for 30 minutes 37oC. The Intracellular pH Calibration Buffer Kit  (Cat#21135) was used to clamp the intracellular pH with extracellular buffers at pH 4.5 to 8.0. Averages of 4 data points were plotted and a 4-parameter trendline was fitted to get the pH standard curve from 6 to 8.
Standard curve created using BCFL, AM (Cat# 21190) and BCECF,AM (Cat#21202) with Intracellular pH Calibration Buffer Kit. Hela cells were incubated with 5 uM BCFL, AM for 30 minutes 37oC. The Intracellular pH Calibration Buffer Kit  (Cat#21135) was used to clamp the intracellular pH with extracellular buffers at pH 4.5 to 8.0. Averages of 4 data points were plotted and a 4-parameter trendline was fitted to get the pH standard curve from 6 to 8.
Standard curve created using BCFL, AM (Cat# 21190) and BCECF,AM (Cat#21202) with Intracellular pH Calibration Buffer Kit. Hela cells were incubated with 5 uM BCFL, AM for 30 minutes 37oC. The Intracellular pH Calibration Buffer Kit  (Cat#21135) was used to clamp the intracellular pH with extracellular buffers at pH 4.5 to 8.0. Averages of 4 data points were plotted and a 4-parameter trendline was fitted to get the pH standard curve from 6 to 8.
The pH dependent standard curve of BCFL and BCECF with various pH buffers. Averages of 3 data points were plotted and a 4-parameter trendline was fitted to get the pH standard curve.
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Additional ordering information
Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight~600
SolventDMSO
Spectral properties
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

OverviewpdfSDSpdfProtocol


Molecular weight
~600
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. However, all the commercial BCECF AM is a complex mixture of at least three isomers with different ratios from batch to batch, complicating the BCECF applications. BCFL is developed to overcome this isomer difficulty associated with BCECF AM. As BCECF, BCFL exhibits a pH-dependent dual excitation, essentially identical to BCECF. It has pKa of ~7.0, identical to BCECF too. As with BCECF, the dual excitation spectrum of BCFL with an isosbestic point at 454 nm should make BCFL a good excitation-ratiometrie pH indicator. BCFL ratiometric imaging makes intracellular pH determination essentially independent of several variable factors, including dye concentration, path length, cellular leakage and photobleaching rate. BCFL, AM is a single isomer, making the pH measurement much more reproducible than the BCECF, AM, which is consisted of quite a few different isomers.

Platform


Fluorescence microscope

ExcitationSingle: FITC, Ratio: AmCyan/FITC
EmissionSingle: FITC, Ratio: AmCyan/FITC
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation430, 505
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.

RatioWorks™ BCFL AM Stock Solution
Prepare a 10 to 20 mM stock solution of RatioWorks™ BCFL AM in high-quality, anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

RatioWorks™ BCFL AM Working Solution
On the day of the experiment, either dissolve RatioWorks™ BCFL AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a RatioWorks™ BCFL 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 RatioWorks™ BCFL 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 RatioWorks™ BCFL 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.  

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)504
Emission (nm)527

Images


Citations


View all 3 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