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

Dette arbeidet ble støttet av en American Heart Association Fellowship Award (16POST29950007) til FL, en trening bevilgning fra National Institutes of Health (NIH T32 HL069765) til BLD, en American Heart Association Fellowship Award (14POST20420025) til MA Saleh, og en NIH K08 award (HL121671) til MSM. MSM er også støttet av en forskningsstipend fra Gilead Sciences, Inc.

Vanderbilt University Institutional Animal Care og bruk komité har godkjent prosedyrene som er beskrevet her. Mus er plassert og omsorg i samsvar med Guide for omsorg og bruk av forsøksdyr (National Academies Press. Revidert 2010). 1. Isolering av Aorta Drenering lymfeknuter, Nyre og Aorta fra Mus <ol><li> Avlive mus ved CO 2 innånding. Spray brystet med 70% etanol og nøye åpne huden og brystveggen med saks for å avsløre hjertet. </li><li> For å perfuse vaskulaturen, utføre et lite snitt i ...

<ol>
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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...

Nyere bevis viser at det adaptive immunsystemet, spesielt T-lymfocytter, spiller en avgjørende rolle i utviklingen av en rekke kardiovaskulære sykdommer 1,2,3,4,5. For eksempel, i modellen av angiotensin II-indusert hypertensjon, en akkumulering av T-celler i skip og nyrene hos mus er blitt beskrevet seks. Den vaskulære opphopning er hovedsakelig i adventitia og perivaskulær fett. I nyrene, T-celler akkumuleres i både medulla og nyrebarken. Avhengig av hvilken undergruppe er involvert, er disse T-cellene gir opphav til forskjellige cytokiner som kan påvirke vaskulær og nyrefunksjon og føre til utvikling av patologi (gjennomgått av McMaster et al. 6). CD4 + T-hjelper-lymfocytter kan deles inn i flere undergrupper: T-hjelper 1 (Th1), Th2, Th9, Th17, Th22, T regulatoriske (treg) celler, og T-hjelper follikulær (TFH) celler basert på deres funksjoner og signatur cytomegaloviruskines 7. På samme måte kan CD8 + cytotoksiske T-celler kan klassifiseres som TC1, TC2, Tc17 eller TC9 8. Det er også doble negative T-celler (dvs. celler som ikke uttrykker CD4 eller CD8 T-cellemarkører). En undergruppe av disse cellene har en alternativ gamma delta T-celle-reseptor (i stedet for de klassiske alfa- og beta-reseptorer) og er derfor referert til som gamma delta T-celler. Den multiparametriske analyse ved flowcytometri av overflatemarkør, cytokin og transkripsjonsfaktor som utgjør den beste tilnærming for å identifisere disse celler. Selv om denne metoden er mye brukt innen immunologien, er det mindre godt beskrevet i faste organer og i innstillingen av kardiovaskulære sykdommer. Historisk ble identifisering av lymfocytter i vev er begrenset til immunhistokjemi eller RT-PCR-metoder. Selv immunhistokjemi og immunfluorescens er kraftige metoder for å bestemme vevet distribusjon av et antigen av interest, de er utilstrekkelig for å fenotypisk identifisere undergrupper involvert. I tillegg, mens RT-PCR-analyse er nyttig for å detektere mRNA ekspresjon av antigener, cytokiner eller transkripsjonsfaktorer, det tillater ikke påvisning av multiple proteiner samtidig på nivået av individuelle celler. Ankomsten av flowcytometri, spesielt kombinert med intracellulær farging for å påvise cytokiner og transkripsjonsfaktorer, gir etterforskere med en kraftfull teknikk som gjør identifisering og kvantifisering på celle nivå av immuncelle undergrupper i solide organer. Vi har optimalisert en intracellulær farging assay for å identifisere ved hjelp av strømningscytometri de store T-celle-undersett til stede i muse-nyre, aorta og aorta drenerende lymfeknuter i en modell av angiotensin II-indusert hypertensjon. Optimaliseringen av hvert trinn: vevsfordøyelse, ex vivo-aktivering, permeabiliseringen, og overflaten og intracellulære fargingsresultater i et høyt reproduser analyse som kan anvendes for andre kardiovaskulære og renale sykdomsmodeller.

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			<td height="21" style="height:21px;">1X Red blood cell lysis buffer</td>
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			<td>Miltenyi Biotec</td>
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			<td height="21" style="height:21px;">Cell activation cocktail (with Brefeldin A)</td>
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			<td height="21" style="height:21px;">123 count eBeads</td>
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			<td height="21" style="height:21px;">CD45 AmCyan (clone 30-F11)</td>
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			<td height="21" style="height:21px;">CD3 PerCP-Cy5.5 (clone 17A2)</td>
			<td>BioLegend</td>
			<td>100218</td>
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			<td height="21" style="height:21px;">IL-17A FITC (clone TC11-18H10.1)</td>
			<td>BioLegend</td>
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			<td height="21" style="height:21px;">IL-17F APC (clone 9D3.1C8)</td>
			<td>BioLegend</td>
			<td>517004</td>
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			<td>560181</td>
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			<td>eBioscience</td>
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			<td height="21" style="height:21px;">T-bet PE-Cy7 (clone 4B10)</td>
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			<td height="21" style="height:21px;">IFN&#947; FITC (clone XMG1.2)</td>
			<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.

Protokollen er beskrevet tillater identifisering av overflate og intracellulære markører i T-celler isolert fra murine nyre, aorta og aorta drenerende lymfeknuter i en modell av angiotensin II-indusert hypertensjon. Representative resultater er presentert nedenfor. Figur 1 viser portstyringsstrategien anvendt for å identifisere T-celle-populasjonen i en enkelt cellesuspensjon fremstilt fra aorta fra et WT m...

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

Denne artikkelen gir detaljert metode for å identifisere og kvantifisere funksjonelle T lymfocytt undergrupper stede i murine nyre, aorta og lymfeknuter ved intracellulær farging og flowcytometri. Modellen av angiotensin II indusert hypertensjon ble valgt til å forklare, steg-for-steg, prosedyrer og grunnleggende prinsipper for flowcytometri og intracellulær farging.

Intracellulær Farging og flowcytometri for å identifisere Lymfocytt Delsett i Murine Aorta, Nyre og lymfeknuter i en 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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