Name: intracellular staining and flow cytometry to identify lymphocyte subsets within murine aorta, kidney and lymph nodes in a model of hypertension
Uploaded: 2017-01-28T13:00:00
Description: Watch this Scientific Journal Video about Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension at JoVE.com

Dit werk werd ondersteund door een American Heart Association Fellowship Award (16POST29950007) naar Florida, een opleiding subsidie ​​van de National Institutes of Health (NIH T32 HL069765) naar BLD, een American Heart Association Fellowship Award (14POST20420025) naar MA Saleh, en een NIH K08 award (HL121671) naar MSM. MSM wordt ook ondersteund door een onderzoek subsidie ​​van Gilead Sciences, Inc.

Vanderbilt University Institutional Animal Care en gebruik Comite heeft de hierin beschreven procedures goedgekeurd. Muizen worden gehuisvest en verzorgd in overeenstemming met de Gids voor de Zorg en gebruik van proefdieren (National Academies Press. Herziene 2010). 1. Isolatie van de Aorta drainerende lymfeklieren, nieren en Aorta van Muizen <ol><li> Euthanaseren de muizen door CO 2 inhalatie. Spray de borst met 70% ethanol en voorzichtig de huid en de borstwand met een schaar om het hart bloot te...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+17+promotes+angiotensin+II-induced+hypertension+and+vascular+dysfunction.">Interleukin 17 promotes angiotensin II-induced hypertension and vascular dysfunction.</a> Hypertension. 55 (2), 500-507 (2010).</li><li>Laroumanie, F., Douin-Echinard, V., Pozzo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T+Cells+Promote+the+Transition+From+Hypertrophy+to+Heart+Failure+During+Chronic+Pressure+Overload.">CD4+ T Cells Promote the Transition From Hypertrophy to Heart Failure During Chronic Pressure Overload.</a> Circulation. 129 (21), 2111-2124 (2014).</li><li>Guzik, T. J., Hoch, N. E., Brown, K. 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The protocol described herein has been optimized to properly identify T cell subsets present within murine kidneys, aorta and lymph nodes. This protocol can be easily adapted to examine other immune cell subsets such as B lymphocytes and innate immune cells and can be modified to include other tissue types. The digestion step is critical and has to be modified and optimized for each tissue9. A prolonged digestion step or the use of an inappropriate enzyme can affect the stability of antigen expression. Similar...

Recent bewijs toont aan dat het adaptieve immuunsysteem, met name T-lymfocyten een cruciale rol spelen bij de ontwikkeling van verscheidene cardiovasculaire aandoeningen 1,2,3,4,5. Bijvoorbeeld, in het model van angiotensine II geïnduceerde hypertensie, een accumulatie van T-cellen in de bloedvaten en nieren van muizen is beschreven 6. De vasculaire accumulatie overwegend adventitia en de perivasculaire vet. In de nier T-cellen ophopen in zowel de medulla en renale cortex. Afhankelijk van welke subset betrokken, deze T-cellen geven aanleiding tot verschillende cytokines die invloed kunnen hebben vasculaire en renale functie en leiden tot de ontwikkeling van de pathologie (beoordeeld door McMaster et al. 6). CD4 + T-helper lymfocyten kunnen worden onderverdeeld in verschillende subgroepen: T helper 1 (Th1), Th2, Th9, Th17, Th22, regulatoire T (Treg) cellen, folliculaire en T helper (Tfh) cellen op basis van hun functie en handtekening cytokines 7. Evenzo kunnen CD8 + cytotoxische T-cellen worden ingedeeld Tc1, Tc2, Tc17 of TC9 8. Er zijn ook een dubbele negatieve T-cellen (dwz cellen die de CD4 of CD8 T-cel markers tot expressie brengen). Een subgroep van deze cellen bezitten een alternatieve gamma delta T-celreceptor (in plaats van de klassieke alfa- en beta-receptoren) en derhalve als gamma delta T-cellen genoemd. Het multiparameter analyse met flowcytometrie van oppervlaktemerker, cytokine- en transcriptiefactor vormt de beste manier om deze cellen te identificeren. Hoewel deze methode op grote schaal wordt gebruikt op het gebied van de immunologie is het minder goed beschreven in vaste organen en bij de vaststelling van hart- en vaatziekten. Historisch gezien is de identificatie van lymfocyten in weefsel beperkt tot immunohistochemie en RT-PCR benadering. Hoewel immunohistochemie en immunofluorescentie zijn krachtige methoden om de weefselverdeling van een antigeen int bepalenerest, ze zijn ontoereikend om de subsets betrokken fenotypisch identificeren. Bovendien, terwijl de RT-PCR is handig om mRNA expressie van antigenen, cytokinen of transcriptiefactoren sporen, het niet de detectie van meerdere eiwitten gelijktijdig kunnen op het niveau van individuele cellen. De komst van flowcytometrie, vooral in combinatie met intracellulaire kleuring cytokinen en transcriptiefactoren detecteren, geeft onderzoekers een krachtige techniek die de identificatie en kwantificering toelaten aan de enkele cel niveau van immuuncelsubklassen in vaste organen. We hebben een intracellulaire kleuring assay te bepalen met stroomcytometrie grote T cel subsets aanwezig in nieren van muizen, aorta en aorta drainerende lymfeklieren in een model van angiotensine II geïnduceerde hypertensie geoptimaliseerd. Optimalisering van elke stap: weefsel spijsvertering, ex vivo activering, permeabilisatie en oppervlak en intracellulaire kleuring leidt tot een zeer reproduceerbaar test die kan worden toegepast op andere cardiovasculaire en renale ziekte modellen.

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			<td>BD Biosciences</td>
			<td>557724</td>
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intracellular staining and flow cytometry to identify lymphocyte subsets within murine aorta, kidney and lymph nodes in a model of hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

Het protocol beschreven maakt de identificatie van oppervlakte- en intracellulaire merkers in T-cellen geïsoleerd uit muizen nieren, aorta en aorta drainerende lymfeklieren in een model van angiotensine II geïnduceerde hypertensie. Representatieve resultaten worden hieronder weergegeven. Figuur 1 toont de gating strategie gebruikt om de T-cel populatie in slechts één enkele celsuspensie bereid uit de aorta...

Watch this Scientific Journal Video about Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension at JoVE.com

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

Dit artikel bevat gedetailleerde methodologie voor het identificeren en kwantificeren van functionele T-lymfocyten subsets in muizen nieren, aorta en de lymfeklieren aanwezig door intracellulaire kleuring en flowcytometrie. Het model van angiotensine II geïnduceerde hypertensie werd gekozen uitleggen, stap voor stap, de procedures en de grondbeginselen van flowcytometrie en intracellulaire kleuring.

Intracellulaire kleuring en flowcytometrie om lymfocyten in muizen Aorta, nieren en lymfeklieren te identificeren in een Model of Hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

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