Name: measuring calpain activity in fixed and living cells by flow cytometry
Uploaded: 2010-07-08T17:15:00
Description: Watch this Scientific Journal Video about Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry at JoVE.com

Supported by grants from the Canadian Institutes of Health Research, The Leukemia and Lymphoma Society of Canada and the Lymphoma Foundation of Canada.

Prepare Reagents
Note: All PBS contains Ca2+ and Mg2+
<ol>
 <li>Detection Reagent 
 Add 20&mu;M 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.</li>
 <li>1% Paraformaldehyde (Fixed Cells) 
 Dilute 4% paraformaldehyde stock solution in room temperature PBS. Aliquot 1mL 1% paraformaldehyde to each FACS tube. Three FACS t...

<ol>
	<li>Goll, D. E., Thompson, V. F., Li, H., Wei, W., Cong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Calpain System. Physiol. Rev. 83, 731-801 (2003).</li><li>Niapour, M., Berger, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+Cytometric+Measurement+of+Calpain+Activity+in+Living+Cells.">Flow Cytometric Measurement of Calpain Activity in Living Cells.</a> Cytometry A. 71A, 475-485 (2007).</li><li>Leloup, L., Daury, L., Maz&#233;res, G., Cottin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+ERK/MAP+kinase+signalling+pathway+in+milli-calpain+activation+and+myogenic+cell+migration.">Involvement of the ERK/MAP kinase signalling pathway in milli-calpain activation and myogenic cell migration.</a> The International Journal of Biochemistry and Cell Biology. 39, 1177-1189 (2007).</li></ol>

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...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>PBS BOC-LM-CMAC 4% paraformaldehyde</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td> Dissolve 4g paraformaldehyde in 100mL PBS. In fume hood, heat sample with stirring until clear. Let cool then store at 4&deg;C</td>
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<td>PD150606</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>Use freshly reconstituted PD150606 for maximal calpain inhibition.</td>
</tr>
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<td>PD98059</td>
<td>&nbsp;</td>
<td>Cell Signaling Technology</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>AlignFlow, AlignFlow plus fluorescent beads FACS tubes</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>LSR II</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>&nbsp;</td>
<td> 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. </td>
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measuring calpain activity in fixed and living cells by flow cytometry

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.

Watch this Scientific Journal Video about Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry at JoVE.com

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

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

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

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