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RatioWorks™ BCFL, AM
Superior replacement for BCECF
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.
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.
protocol
Protocol
sds
SDS
COA
CatalogSize
Price
Quantity
211901 mg
Price
 
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
Instrument settings
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
Documents
Protocol
Safety Data Sheet
Certificate of Analysis
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Page updated on October 21, 2025