RatioWorks™ BCFL, AM *Superior replacement for BCECF*

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1 mg 21190 $145

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Ex/Em (nm)503/528
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsRatio-Imaging Microscopy, Fluorescence Microscope, Fluorescence Microplate Reader, Flow Cytometer
Category Cell Biology
pH and Ion Indicators
Related Cell Metabolism
Secondary Reagents
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.

Common stock solution preparation

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

Molarity calculator

Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
/ = x =


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This protocol only provides a guideline, and should be modified according to your specific needs.

1. Prepare cells as desired. For example, plate adherent cells overnight in growth medium at 40,000 to 80,000 cells/well/100µL for  

       96-well or 10,000 to 20,000 cells/well/25µL for 384-well plates.

Note:  Each cell line should be evaluated on an individual basis to determine the optimal cell density.

2. Prepare RatioWorks™ BCFL, AM dye-loading solution:

2.1    Prepare a 10 to 20 mM stock solution of RatioWorks™ BCFL, AM in high-quality, anhydrous DMSO. The stock solution should be used promptly. Any unused solution need to be aliquoted and refrozen at < -20 oC.

Note: Avoid repeated freeze-thaw cycles, and protect from light.

2.2    Prepare a 5-50 uM RatioWorks™ BCFL, AM dye-loading solution in Hanks and 20 mM Hepes buffer (HHBS).


3.  Run pH Assay

3.1    Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) RatioWorks™ BCFL, AM dye-loading solution into the cell plate (from Step 2.3).

Note:  It is important to replace the growth medium with HHBS buffer (100 µL/well for 96-well plate or 25 µL/well for 384-well plate before dye-loading) if your compounds interfere with the serum.

3.2    Incubate the dye-loading plate at cell incubator for 30 to 60 minutes.

3.3    Wash and replace the dye-loading solution with HHBS.

3.4    Prepare the compound plates by using HHBS or your desired buffer.

3.5    Run the pH assay by monitoring the fluorescence at Ex/Em = 490/535 nm (cut off at 515 nm) or 505/535 nm and 430/535 nm (cut off at 515 nm) for ratio measurements. The compound addition is 50 µL/well (96-well plate) or 25 µL/well (384-well plate).

Note: The assay should be complete within 3 to 5 min after compound addition, however a minimum of 8 min data collection are recommended for during assay development.

References & Citations

The F0F1 ATP synthase regulates human neutrophil migration through cytoplasmic proton extrusion coupled with ATP generation
Authors: Jun Gao, Tian Zhang, Zhanfang Kang, Weijen Ting, Lingqing Xu, Dazhong Yin
Journal: Molecular Immunology (2017): 219--226

Oxidative Stress-Activated NHE1 Is Involved in High Glucose-Induced Apoptosis in Renal Tubular Epithelial Cells
Authors: Yiqing Wu, Min Zhang, Rui Liu, Chunjie Zhao
Journal: Yonsei Medical Journal (2016): 1252--1259

Additional Documents

Safety Data Sheet (SDS)

1. Physiological Probes & Assay Kits

Application Notes
1. AssayWise Letters 2013, Vol 2(2)

Certificate of Analysis