Intracellular pH Assay Kit (ab228552)
Key features and details
- Assay type: Cell-based (quantitative)
- Detection method: Fluorescent
- Platform: Microplate reader
- Sample type: Adherent cells, Suspension cells
Overview
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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.
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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.
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Platform
Microplate reader
Properties
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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
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Carbachol dose response in CHO-M1 cells
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).