Creative Biolabs

pH Indicators

pH Indicators

pH Indicators

Changes in cytosolic intracellular pH play a central role in many physiological processes such as muscle contraction, nerve-impulse propagation, and cell signaling. Unlike intracellular free Ca2+ concentrations, which can rapidly change by perhaps 100-fold, intracellular pH varies by only fractions of a pH unit, and such changes may occur quite slowly. Creative Biolabs now offers a range of novel indicators enabling you to track pH in the cytosol and in particular organelles.

pH Indicators at a Glance


BCECF is the most popular fluorescent pH sensor. Its pKa of 6.98 is ideal because all cells have intracellular pH near 7.0, with physiological changes of no more than a few tenths of a pH unit. At low pH, the dye is weakly fluorescent but becomes more fluorescent with increasing pH. The excitation spectrum of the dye undergoes a slight shift with pH change, while the wavelength of the emission maximum remains unchanged. BCECF has an excitation spectrum with a maximum at 507 nm and an isoexcitation point at 438 nm. Intracellular pH can therefore be estimated by measuring the ratio of the fluorescence intensities at 500 and 450 nm. Commercial BCECF indicators are available in membrane-impermeant free acid and cell-permeable AM ester forms.

  • Flubida-2 pH Indicators

Flubida-2 has been used to detect pH at a specific site in a cell such as cell organelles by directing the probe to where avidin-chimera proteins are located. The probe is a conjugate of biotin and fluorescein diacetate, which is non-fluorescent until the probe has entered the cells and is hydrolyzed by cellular esterases. Flubida-2 is membrane-permeable and can be delivered into cells via simple incubation.

  • Carboxyfluorescein pH Indicators

With a pKa of 6.5, Carboxyfluorescein (FAM) can be used as a pH indicator or cellular tracer. Its excitation spectrum and fluorescence response to pH are similar to those of BCECF. FAM is also available in membrane permeable diacetate form. 5-(and-6)-Carboxy-2',7'-dichlorofluorescein is similar to FAM with a pKa of 4.8. It is useful for detecting pH in more acidic environment.

Tracking pH in vesicles

Tracking internalization of fluorescent dextran is a routine method for analyzing pH changes in cellular compartments. Dextran conjugates of pHrodo dyes provide the most complete solution by allowing discrimination of vesicles from early endosomes to lysosomes. pHrodo dyes are non-fluorescent at neutral pH and provide signal intensity modulation from pH 2 to pH 9 with a choice of fluorescent wavelengths. By conjugating pHrodo Red together with Rhodamine Green onto a 10,000 MW dextran, the dual-labeled probe also provides a reference signal for tracking.

How to Select the Optimal pH Indicator for your Experiments?

Fluorescein derivatives are available as single isomer and mixed isomers. The isomers have the same spectral properties. Single isomer may be preferable for certain coupling reactions but mixed isomers are suitable for most applications.

Membrane-Permeant pH Indicators Membrane-Impermeant pH Indicators
BCECF AM Ester pKa 6.97 BCECF, free acid pKa 6.97
5-Carboxyfluorescein diacetate (single isomer)  pKa 6.5 5-Carboxyfluorescein (5-FAM, single isomer)  pKa 6.5
6-Carboxyfluorescein diacetate (single isomer) 6-Carboxyfluorescein (6-FAM, single isomer)
5-(and-6)-Carboxyfluorescein diacetate (mixed isomers) 5-(and-6)-Carboxyfluorescein (5(6)-FAM, mixed isomers)
5-(and-6)-Carboxyfluorescein diacetate, succinimidyl ester Flubi-2
Flubida-2 5-Carboxy-2',7'-dichlorofluorescein pKa 4.8
5-(and-6)-Carboxy-2',7'-dichlorofluorescein diacetate pKa 4.8 5-(and-6)-Carboxy-2',7'-dichlorofluorescein
5-(and-6)-Carboxy-2',7'-dichlorofluorescein diacetate, succinimidyl ester 5-Carboxy-2',7'-dichlorosulfonefluorescein pKa 4
  Cytoplasmic pH Indicators Vesicle pH Indicators
Standard filter set FITC; TRITC; Requires excitation ratio to quantify pH FITC; TRITC; Requires excitation ratio to quantify pH
Fluorophore pHrodo Green; pHrodo Red; BCECF pHrodo Green; pHrodo Red; BCECF
Cell permeant or impermeant? Permeant Impermeant
Usage notes Load in cell media Loaded by endocytosis, microinjection, patch pipette of pinocytic loading agent
Platforms Flow cytometer, microplate reader, imaging Flow cytometer, microplate reader, imaging

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