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

Ondersteund door subsidies van de Canadese Institutes of Health Research, de leukemie en lymfoom Society van Canada en de Stichting Lymfoom van Canada.

Reagentia voor te bereiden Opmerking: Alle PBS bevat Ca 2 + en Mg 2 + <ol><li> Detectie Reagens Voeg 20μM 7-amino-4-chloormethyl coumarine, t-BOC-Leucine-methionine amide (BOC-LM-CMAC) op kamertemperatuur PBS. Elke cel schorsing vereist 2 ml detectie reagens. </li><li> 1% Paraformaldehyde (vaste cellen) Verdunnen 4% paraformaldehyde stockoplossing bij kamertemperatuur PBS. Aliquot 1 ml 1% paraformaldehyde aan elke FACS buis. Drie FACS buizen zijn nodig ...

<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>

De calpaïne activiteit in de vaste en levende cellen 32Dkit is bepaald in deze video protocol. Behandeling van de cel lijn met de calpaïne remmer PD150606 resulteerde in complete remming van calpaïne activiteit in de cellen. Daarnaast is behandeling met de remmer MEK1 PD98059 resulteerde in complete remming van calpaïne activiteit in de cellen. ERK is een belangrijke activator van calpaïne-2 3. Deze resultaten suggereren dat calpaïne-2 activiteit is overheersend in de 32Dkit cellen. Huidige werk in ons ...

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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>
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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 zijn alomtegenwoordig intracellulaire, calcium-gevoelige, neutrale cysteine ​​proteases<sup&gt; 1</sup&gt;. Calpains spelen een cruciale rol in vele fysiologische processen, met inbegrip van seinen, cytoskelet remodeling, regulatie van genexpressie, apoptose en voortgang van de celcyclus<sup&gt; 1</sup&gt;. Calpains zijn betrokken bij tal van pathologieën waaronder spierdystrofieën, kanker, diabetes, de ziekte van Alzheimer en multiple sclerose<sup&gt; 1</sup&gt;. Calpaïne regelgeving is complex en onvolledig begrepen. mRNA en eiwit-concentraties zijn gecorreleerd slecht met de activiteit, beperking van het gebruik van gen-of eiwitexpressie technieken om calpaïne activiteit te meten. Deze video protocol details ontwikkelde een flowcytometrische analyse in ons laboratorium voor het meten van calpaïne activiteit in vaste en levende cellen. Deze methode maakt gebruik van de fluorescent substraat BOC-LM-CMAC, die specifiek wordt gesplitst door calpaïne, op maat calpaïne activiteit.<sup&gt; 2</sup&gt; In deze video, is calpaïne activiteit in de vaste en levende muizen 32Dkit leukemie cellen, alleen of als onderdeel van een splenocyten bevolking gemeten met behulp van een LSRII (BD Bioscience). 32Dkit cellen blijken te hebben verheven activiteit in vergelijking met normale splenocyten.

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

Dit artikel zal detail het protocol voor het meten van calpaïne activiteit in de vaste en levende cellen met behulp van flow cytometrie.

Het meten van calpaïne Activiteit in vaste en levende cellen door flowcytometrie

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

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