Protocol

Calcium Signaling Protocols

Volume 937 of the series Methods in Molecular Biology pp 163-174

Date:

Measurement of Phospholipase C by Monitoring Inositol Phosphates Using [3H]Inositol Labeling Protocols in Permeabilized Cells

  • Alison SkippenAffiliated withDepartment of Neuroscience, Physiology, and Pharmacology, University College London
  • , Philip SwigartAffiliated withDepartment of Neuroscience, Physiology, and Pharmacology, University College London
  • , Shamshad CockcroftAffiliated withDepartment of Neuroscience, Physiology, and Pharmacology, University College London Email author 

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Abstract

Data on the production of inositol phosphates is a useful complement to measurements of intracellular Ca2+. The basic principle is labeling of the inositol lipids by growing the appropriate cell line in culture in the presence of [3H]inositol for 2–3 days to reach labeling equilibrium. Lithium ions at 10 mM inhibits the degradation of inositol phosphates to free inositol and is used to trap the inositol in the inositol polyphosphate forms. Inositol phosphates can be separated with ease from free inositol by using anion exchange chromatography. A method capable of easily processing approximately 40–60 samples in a single day is presented.

Key words

Phospholipase C [3H]Inositol phosphates Permeabilized cells