Overview

  • Product name

    Intracellular pH Assay Kit
  • Detection method

    Fluorescent
  • Sample type

    Adherent cells, Suspension cells
  • Assay type

    Cell-based (quantitative)
  • Product overview

    The Intracellular pH Assay Kit (ab228552) utilizes a proprietary fluorescent indicator for measuring relative intracellular pH changes. It is an homogeneous, kinetic, live-cell fluorescent assay that utilizes either a standard procedure or acid-load procedure. The standard protocol is designed for measuring the therapeutic targets of interest with a decrease in intracellular pH upon treatment. The 'Acid-Load' procedure is designed to measure the increase of intracellular pH associated with changes in cellular metabolism due to GPCR activation or growth factor activity. 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.

  • Notes

    Intracellular pH change 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.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 1000 tests
    10X Pluronic F127 Plus 1 x 10ml
    50 mM probenecid 1 x 10ml
    BCFL, AM 1 vial
    HHBS 1 x 100ml

Images

  • CHO-M1cells were seeded overnight in 60,000 cells per 100 µL per well in a 96-well black wall/clear bottom Costar plate. The growth medium was replaced with 50 µL/well of RatioWorks™ BCFL, AM dyeloading solution for 37°C for 1 hour, follow by 15 minutes incubation with 5 µL/well of 220 mM NH4Cl. Carbachol (200µL/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations. The fluorescent signal was generated using Ex/Em = 490/535 nm (cut off at 525 nm).

Protocols

References

ab228552 has not yet been referenced specifically in any publications.

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