Calcium Signaling Protocols pp 163-174

Part of the Methods in Molecular Biology book series (MIMB, volume 937)

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

  • Alison Skippen
  • Philip Swigart
  • Shamshad Cockcroft
Protocol

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 

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Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Alison Skippen
    • 1
  • Philip Swigart
    • 1
  • Shamshad Cockcroft
    • 1
  1. 1.Department of Neuroscience, Physiology, and PharmacologyUniversity College LondonLondonUK

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