Measuring calpain activity in fixed and living cells by flow cytometry.
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.
Farr
Christina
C
Immunology, University of Toronto, Canada.
Berger
Stuart
S
D000179:Acrylates / Q000494:pharmacology; D000818:Animals; D002154:Calpain / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D045744:Cell Line, Tumor; D003374:Coumarins / Q000737:chemistry; D004151:Dipeptides / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D007938:Leukemia / Q000201:enzymology; D051379:Mice; D013154:Spleen / Q000166:cytology / Q000201:enzymology
D000179:Acrylates; D003374:Coumarins; D004151:Dipeptides; C100442:PD 150606; C522288:t-BOC-leucine-methionine amide-CMAC; D002154:Calpain