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

Supportato da sovvenzioni da parte del Canadian Institutes of Health Research, The Leukemia and Lymphoma Society of Canada e la Fondazione Linfoma del Canada.

Preparare Reagenti Nota: Tutti i PBS contiene Ca 2 + e Mg 2 + <ol><li> Rilevazione dei reagenti Aggiungi 20μM 7-ammino-4-clorometil cumarina, t-BOC-Leucina-metionina ammide (BOC-LM-CMAC) PBS a temperatura ambiente. Ogni sospensione cellulare richiederà reagente 2 ml di rilevamento. </li><li> 1% paraformaldeide (Cellule fisso) Diluire soluzione al 4% paraformaldeide magazzino a temperatura ambiente PBS. Aliquota paraformaldeide 1 ml 1% a ciascuna provet...

<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>

L&#39;attività calpaina in cellule 32Dkit fisse e vita è stato determinato in questo protocollo video. Il trattamento della linea cellulare con l&#39;inibitore della calpaina PD150606 portato a completa inibizione dell&#39;attività calpaina nelle cellule. Inoltre, il trattamento con l&#39;inibitore MEK1 PD98059 portato a completa inibizione dell&#39;attività calpaina nelle cellule. ERK è un importante attivatore di calpaina-2 3. Questi risultati suggeriscono che l&#39;attività calpaina-2 è predominante...

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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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<tr>
<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 sono onnipresenti intracellulari, calcio-sensibili, la cisteina proteasi neutre<sup&gt; 1</sup&gt;. Calpains svolgono un ruolo cruciale in molti processi fisiologici, tra cui segnalazione, rimodellamento del citoscheletro, regolazione dell&#39;espressione genica, l&#39;apoptosi e la progressione del ciclo cellulare<sup&gt; 1</sup&gt;. Calpains sono stati coinvolti in molte patologie tra cui le distrofie muscolari, cancro, diabete, morbo di Alzheimer e la sclerosi multipla<sup&gt; 1</sup&gt;. Calpaina regolamento è complesso e non completamente compreso. livelli di mRNA e proteine ​​correlate con l&#39;attività poco, limitando l&#39;uso di tecniche di espressione dei geni o proteine ​​per misurare l&#39;attività calpaina. Questo il video dettagli protocollo un test citometria a flusso sviluppato nel nostro laboratorio per la misurazione dell&#39;attività calpaina in cellule fisse e viventi. Questo metodo utilizza il substrato fluorescente BOC-LM-CMAC, che viene scissa in particolare da calpaina, per misurare l&#39;attività calpaina.<sup&gt; 2</sup&gt; In questo video, attività calpaina nelle cellule 32Dkit fisso e vive leucemia murina, da soli o come parte di una popolazione splenocyte viene misurata utilizzando un LSRII (BD Bioscience). Le cellule sono 32Dkit dimostrato di avere attività elevata rispetto a splenociti normali.

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

Questo articolo dettaglio il protocollo per la misurazione dell&#39;attività calpaina in cellule fisse e vivere in citometria a flusso.

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

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