Farr Christina, Berger Stuart
Immunology, University of Toronto, Canada.
J Vis Exp. 2010 Jul 8(41):2050. doi: 10.3791/2050.
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.
钙蛋白酶是普遍存在于细胞内的、对钙敏感的中性半胱氨酸蛋白酶。钙蛋白酶在许多生理过程中发挥关键作用，包括信号传导、细胞骨架重塑、基因表达调控、细胞凋亡和细胞周期进程。钙蛋白酶与多种病理学相关，包括肌肉萎缩症、癌症、糖尿病、阿尔茨海默病和多发性硬化症。钙蛋白酶的调节很复杂且尚未完全了解。mRNA和蛋白质水平与活性的相关性很差，限制了使用基因或蛋白质表达技术来测量钙蛋白酶活性。本视频方案详细介绍了我们实验室开发的一种用于测量固定细胞和活细胞中钙蛋白酶活性的流式细胞术检测方法。该方法使用荧光底物BOC-LM-CMAC，它被钙蛋白酶特异性切割，以测量钙蛋白酶活性。在本视频中，使用LSRII（BD Bioscience）测量固定的和活的小鼠32 Dkit白血病细胞单独或作为脾细胞群体一部分时的钙蛋白酶活性。结果显示，与正常脾细胞相比，32 Dkit细胞具有更高的活性。
