Protocol

Neutrophil Methods and Protocols

Volume 1124 of the series Methods in Molecular Biology pp 89-105

Date:

Measurement of Phospholipid Metabolism in Intact Neutrophils

  • Susan SergeantAffiliated withDepartment of Biochemistry, Wake Forest University School of Medicine
  • , Linda C. McPhailAffiliated withDepartment of Biochemistry, Wake Forest University School of Medicine

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Abstract

Phospholipid-metabolizing enzymes are important participants in neutrophil signal transduction pathways. The methods discussed herein describe assays for assessing the activities of phospholipase A2 (PLA2), phospholipase C (PLC), phospholipase D (PLD), and phosphoinositide 3-OH-kinase in intact neutrophils. PLA2 activity is measured as the release of radiolabeled arachidonic acid. PLC activity is measured as the accumulation of inositol 1,4,5-trisphosphate (IP3), a water-soluble product, using a commercially available radioreceptor assay kit. PLD activity is measured as the appearance of its radiolabeled products, phosphatidic acid and phosphatidylethanol. PI3-K activity is measured as the appearance of its radiolabeled product, phosphatidylinositol-3,4,5-trisphosphate.

Keywords

Neutrophil Phospholipase Lipid kinase Phospholipid Signal transduction Lipid second messengers