Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

التفاصيل البيبلوغرافية
العنوان: Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry
المؤلفون: Stuart A. Berger, Christina Farr
المصدر: Journal of Visualized Experiments.
بيانات النشر: MyJove Corporation, 2010.
سنة النشر: 2010
مصطلحات موضوعية: Proteases, General Chemical Engineering, Population, General Biochemistry, Genetics and Molecular Biology, Flow cytometry, Mice, Coumarins, Cell Line, Tumor, Splenocyte, medicine, Animals, education, Regulation of gene expression, education.field_of_study, Leukemia, biology, medicine.diagnostic_test, General Immunology and Microbiology, Calpain, General Neuroscience, Dipeptides, Flow Cytometry, Molecular biology, Cell biology, JoVE Immunology, Acrylates, Apoptosis, biology.protein, Intracellular, Spleen
الوصف: Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. 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. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.
تدمد: 1940-087X
DOI: 10.3791/2050-v
URL الوصول: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::6c18b2b05dd1138d6f444412e1897c7d
https://doi.org/10.3791/2050-v
Rights: OPEN
رقم الانضمام: edsair.doi.dedup.....6c18b2b05dd1138d6f444412e1897c7d
قاعدة البيانات: OpenAIRE
الوصف
تدمد:1940087X
DOI:10.3791/2050-v