Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Summary

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Abstract

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.

Protocol

Prepare Reagents

Note: All PBS contains Ca²⁺ and Mg²⁺

1. Detection Reagent
   Add 20μM 7-amino-4-chloromethyl coumarin, t-BOC-Leucine-methionine amide (BOC-LM-CMAC) to room temperature PBS. Each cell suspension will require 2mL detection reagent.
2. 1% Paraformaldehyde (Fixed Cells)
   Dilute 4% paraformaldehyde stock solution in room temperature PBS. Aliquot 1mL 1% paraformaldehyde to each FACS tube. Three FACS tubes are required for each experimental condition (ex. 32Dkit, 32Dkit + PD150606, 32Dkit + PD98059 need 9 FACS tubes)
3. PD150606 (calpain inhi....

Discussion

The calpain activity in fixed and living 32Dkit cells has been determined in this video protocol. Treatment of the cell line with the calpain inhibitor PD150606 resulted in complete inhibition of calpain activity in the cells. In addition, treatment with the MEK1 inhibitor PD98059 resulted in complete inhibition of calpain activity in the cells. ERK is a major activator of calpain-2 ³. These results suggest that calpain-2 activity is predominant in the 32Dkit cells. Current work in our laboratory is investigat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PBS BOC-LM-CMAC 4% paraformaldehyde	Invitrogen		Dissolve 4g paraformaldehyde in 100mL PBS. In fume hood, heat sample with stirring until clear. Let cool then store at 4°C
PD150606	Invitrogen		Use freshly reconstituted PD150606 for maximal calpain inhibition.
PD98059	Cell Signaling Technology
AlignFlow, AlignFlow plus fluorescent beads FACS tubes	Invitrogen
LSR II	BD Biosciences		Supernatant of WEHI-3 cells is used as a source of IL-3 in these experiments. Each collection of WEHI-3 supernatant is titrated to determine the optimal concentration used for 32Dkit cell growth.