Fluorometric Intracellular pH Assay Kit, sufficient for 1000 fluorometric�tests

Application

Fluorometric Intracellular pH Assay Kit has been used to measure the intracellular pH.

Features and Benefits

Superior alternative to BCECF

General description

Intracellular pH changes are implicated in diverse physiological and pathological processes, including cell proliferation, apoptosis, fertilization, malignancy, multidrug resistance, ion transport, lysosomal storage disorders, and Alzheimer′s disease.This kit can be utilized to measure decreases in intracellular pH in cells treated under various conditions using a simple standard protocol. Alternatively, an Acid-Load procedure can be used to measure the increase of intracellular pH associated with changes in cellular metabolism due to GPCR activation, growth factor activity, or other cellular stimulation. With the Acid-Load procedure ammonium chloride solution is added after the fluorescent pH dye is loaded into cells in a minimum volume. This Acid-Loading step is followed by the addition of agonist in a relatively large volume (~4x) of buffer. The sudden volume change initiates an efflux of ammonia (NH3) from the cells causing a rapid decrease in intracellular pH, and thus a decrease in fluorescence signal. The effect of agonist on the subsequent recovery of intracellular pH is measured by the relative fluorescence signal increase.

Principle

The Fluorometric Intracellular pH Assay Kit utilizes a proprietary cell-permeable fluorescent indicator, BCFL-AM, for measuring relative intracellular pH changes. BCFL-AM is a superior alternative to the popular ratiometric pH probe, 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Unlike BCECF, which is a complex and variable mixture of isomers, BCFL-AM is a single isomer species. BCFL-AM has the same spectral responses as BCECF-AM (λ_ex = 490 nm/λ_em = 535 nm for single measurement or λ_ex = 505 nm/λ_em = 535 nm and λ_ex = 430 nm/λ_em = 535 nm for ratio measurement) but enhanced reproducibility. BCFL-AM exhibits pH-dependent dual excitability, pKa ~7.0, and an excitation isosbestic point of 454 nm.

Suitability

This kit can be utilized to measure changes in intracellular pH in cells treated under various conditions.