Measuring calpain activity in fixed and living cells by flow cytometry.

Journal of Visualized Experiments : JoVE
C Farr, Stuart Berger

Abstract

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. In this video, calpain activity in fixed and living murine 32 Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32 Dkit cells are shown to have elevated activity compared to normal splenocytes.

References

Jul 5, 2003·Physiological Reviews·Darrell E GollJinyang Cong
Apr 17, 2007·The International Journal of Biochemistry & Cell Biology·Ludovic LeloupJean-Jacques Brustis
Apr 27, 2007·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Maryam Niapour, Stuart Berger

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Citations

Aug 21, 2012·Neuromolecular Medicine·Sohila ZadranMichel Baudry
Mar 1, 2012·Experimental Hematology·Hongbing LiStuart A Berger
Dec 24, 2013·PLoS Computational Biology·Kieran O'NeillRyan Brinkman
Nov 4, 2017·Cell Adhesion & Migration·Pablo A MendozaVicente A Torres

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