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Flow cytometric measurement of calpain activity in living cells
Author(s) -
Niapour Maryam,
Berger Stuart
Publication year - 2007
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20399
Subject(s) - calpain , proteases , chemistry , flow cytometry , intracellular , microbiology and biotechnology , biochemistry , biology , enzyme
Background: Calpains are intracellular, calcium‐sensitive, neutral cysteine proteases that play crucial roles in many physiological and pathological processes. Calpain regulation is complex and activity is poorly correlated with calpain protein levels. Therefore a full understanding of calpain function requires robust methods for measuring activity. Methods: We describe and characterize a flow cytometric method for measuring calpain activity in live cells. This method uses the BOC‐LM‐CMAC reagent that readily diffuses into cells where it reacts with free thiols to enhance retention. Results: We show that the reagent is cleaved specifically by calpains and follows saturation kinetics. We use the assay to measure calpain activation following PDGF stimulation of rat fibroblasts. We also show that the calpain inhibitor PD150606 inhibits calpain with a K i of 12.5 μM and show that Mek inhibitors PD89059 and U0126 also suppress calpain activity. We also show that the assay can measure calpain activity in subpopulations of cells present in unfractionated cord blood or in HL60 human myelomonocytic leukemia cells. Conclusion: Taken together, these experiments demonstrate that this assay is a reliable and useful method for measuring calpain activity in multiple cell types. © 2007 International Society for Analytical Cytology