Name: isolating and immunostaining lymphocytes and dendritic cells from murine peyer's patches
Uploaded: 2013-03-17T14:00:00
Description: Watch this Scientific Journal Video about Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches at JoVE.com

Vi takker Renjie Song (Wadsworth center Flowcytometri Core) for at få hjælp i celle analyse og Helen Johnson (Wadsworth center Animal Histopatologi Core) til fremstilling af paraffinsnit. Vi takker Dr. Richard A. Cole (Wadsworth center lysmikroskopi Core) for at få hjælp med konfokal mikroskopi og billedsamling. Vi vil gerne anerkende Andy Bentley (Wadsworth Center-billede og Illustration) for at få hjælp med animationer. MDJ er støttet af Life Sciences Research Foundation, Howard Hughes Medical Institute (HHMI) Fellowship. SA er understøttet af en Wadsworth Center-Health Research Inc. murene postdoc-stipendium. Dette arbejde blev støttet delvist af NIH tilskud HD061916 og GM082978.

Dyrene blev opstaldet under konventionelle, specifikke patogenfrie betingelser og blev behandlet i fuld overensstemmelse med Wadsworth Centers Institutional Animal Care og Use Committee (IACUC) retningslinjer. 1. Oral gavage <ol><li> (Valgfrit) gavage musestamme af valg med antigen eller mikrober af interesse ved hjælp af en 22 G x1.5-in. stump-ende fodring nål (Popper Scientific, New Hyde Park, NY). Levering mængder bør ikke overstige 400 pi per mus. </li></ol> 2...

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I denne artikel har vi givet parallelle protokoller til fremstilling PP encellede forberedelserne til flowcytometrisk og funktionel analyse og kryosektioner for immunfarvning. Begge metoder er meget reproducerbar og let tilgængelig, tilvejebragt et flow-cytometer og kryostat er tilgængelige. For første gang efterforskere skal det påpeges, at i forhold til milten, alt celleudbytter fra PPs er forholdsvis beskedne. Alligevel protokollen vi skitsere generelt udbytter mellem 0,8-1,2 x 10 6 totale PP celler fr...

Peyerske plaques (PP) er makroskopiske aggregater af organiserede lymfoide follikler stede i hele tyndtarmen hos mennesker og mus (figur 1) og udgør de primære steder, hvor mucosale immunresponser der indledes over diæt antigener, kommensale bakterier, mikrobielle patogener samt orale vacciner 1-4. I modsætning til andre perifere lymfoide væv såsom mesenteriallymfeknuder, mangler PPs afferente lymfekar. Som sådan, adaptive immunrespons hos PP drives som reaktion på antigener afledt fra det intestinale lumen. Udtagning af prøver af luminale antigener opnås det ved follikel-associerede epitel (FAE), som består af både enterocytter og antigen-sampling celler kendt som M-celler. Under FAE, i sub-epithelial kuppel (SED) region ligger et netværk af dendritiske celler (DC&#39;er) blandet sammen med makrofager, B-celler og CD4 + T-celler 5-9. Kernen i hvert PP lymfoide follicle er follikulære dendritceller (FDC&#39;er) og en B-celle-rige centrale germinal center, flankeret af T-celle-rige interfollicular zoner. Antigen prøveudtagning af PP resulterer i udviklingen af IgA + B-celle plasmablasts og CD4 +-effektor-og memory-celler, at frø den omgivende lamina propria og giver immunitet over for en lang række til mucosale angribere. Dissekere de komplekse immunologiske hændelser, der er forbundet med antigen prøvetagning, behandling og præsentation i PP er en skræmmende opgave, i betragtning af at PP-celler udgør kun en lille brøkdel af de samlede lymfoide celler i tarmslimhinden. Til hjælp i in vitro karakterisering af celler i dette miljø, vi leverer en protokol til fremstilling af total muse PP-celler til flowcytometrisk og funktionel analyse, samt en protokol for at forberede PP kryosektioner for immunfluorescensmikroskopi og immunhistologi. Vores protokol til isolering, karakterisering og immunfarvning af aftalememorandae PP-celler er ikke ny i sig selv, som det fremgår af den kendsgerning, at der er talrige referencer helt tilbage mere end 25 år, der citerer disse teknikker 5,6,9-11. Snarere vores protokol giver en strømlinet (og visuelt) metode for efterforskerne indsamler PPs for første gang. De teknikker, vi beskriver, er let styr og let danner et stort antal celler med&gt; 90% cellelevedygtighed. Den cryosectioning protokol giver meget reproducerbare seriesnit ideelt egnede til immunfluorescensfarvning og konfokal billeddannelse. Desuden er vores protokol supplerer to andre nylige Jupiter artikler. Den første, som Fukuda og kolleger, beskriver anvendelsen af ligerede ileal løkke-assays for at vurdere optagelsen af patogene bakterier ved PP M celler 12. Dels ved Geem og kolleger, beskriver isoleringen og karakteriseringen af DC&#39;er og makrofager fra mus tarmslimhinden, men udelukker udtrykkeligt PPs fra deres analyse 13.

<table border="1"><tbody><tr><td> Item </td><td> Firma </td><td> Kat. # </td><td> Kommentarer (valgfrit) </td></tr><tr><td> OCT-forbindelse </td><td> Tissue-Tek </td><td> 4583 </td><td></td></tr><tr><td> 7x7x5 mm base Molds </td><td> Fisherbrand </td><td> 22-363-552 </td><td></td></tr><tr><td> ImmEdge Pen </td><td> Vector Labs </td><td> H-4000 </td><td></td></tr><tr><td> SuperFrost Plus Slides </td><td> Thermo Scientific </td><td> 4951 </td><td></td></tr><tr><td> Edge Rite Blade </td><td> Thermo Scientific </td><td> 4280L </td><td></td></tr><tr><td> Anti-Mouse CD11c-PE </td><td> eBioscience </td><td> 17-0114-82</td><td></td></tr><tr><td> Anti-Mouse CD45R/B220-APC </td><td> BD Pharmingen </td><td> 553.092 </td><td></td></tr><tr><td> Anti-Mouse CD3-FITC </td><td> BD Pharmingen </td><td> 561.798 </td><td></td></tr><tr><td> Anti-muse-CD4-PE </td><td> BD Pharmingen </td><td> 553.652 </td><td></td></tr><tr><td> Anti-Mouse CD8-PE </td><td> BD Pharmingen </td><td> 553.032 </td><td></td></tr><tr><td> Anti-Mouse CD19-PerCP </td><td> BioLegend </td><td> 115.531 </td><td></td></tr><tr><td> Hanks balancerede saltopløsning (HBSS) uden phenolrødt </td><td> Fisher Scientific </td><td> 14175-079 </td><td></td></tr><tr><td> 70 pm cell strainer </td><td> BD Falcon </td><td&gt; 352350 </td><td></td></tr><tr><td> Spleen Dissociation Medium </td><td> Stem Cell Technologies </td><td> 7915 </td><td></td></tr><tr><td> Gedeserum </td><td> Invitrogen </td><td> 16210-072 </td><td></td></tr><tr><td> Fc Block </td><td> ATCC 2.4.G2 </td><td> HB-197 </td><td> Supes opnået fra cellelinien 2.4.G2 </td></tr><tr><td> Buet Scissor </td><td> FST </td><td> 14.061-09 </td><td></td></tr><tr><td> Kryostat </td><td> Leica </td><td> 3050S </td><td></td></tr><tr><td> FACS Calibur </td><td> BD </td><td></td><td></td></tr><tr><td> Grevinde Cell Counter </td><td> Invitrogen </td><td></td><td></td></tr><tr><td> Hematoxylin </td&gt; <td> Richard Allan </td><td> 7211 </td><td></td></tr><tr><td> Eosin </td><td> Richard Allan </td><td> 71.304 </td><td></td></tr><tr><td> Formalin </td><td> Starplex Scientific </td><td> 3661 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabel 1. Reagenser og udstyr anvendt i denne undersøgelse. </td></tr></tbody></table>

isolating and immunostaining lymphocytes and dendritic cells from murine peyer's patches

Peyerske plaques (PP) er uadskillelige dele af tarmen-associerede lymfoide væv (GALT) og spiller en central rolle i intestinal immunosurveillance og homeostase. Partikelformige antigener og mikrober i det intestinale lumen kontinuerligt samplet af PP M-celler i follicle-associerede epitel (FAE) og transporteres til et underliggende netværk af dendritiske celler (DC&#39;er), makrofager og lymfocytter. I denne artikel beskriver vi protokoller, i hvilke murine PPs er (i) dissocieret til enkeltcelle-suspensioner og underkastet strømningscytometri og (ii) forberedt til cryosectioning og immunfarvning. Til flowcytometri, er PP dissocieret mekanisk og derefter filtreret gennem 70 um membraner til at generere enkeltcelle-suspensioner fri for epitelceller og stort affald. Startende med 20-25 PPs (fra fire mus), den hurtige og reproducerbar metode giver en population af&gt; 2,5 x 10 6 celler med&gt; 90% cellelevedygtighed. For cryosectioning, friskely isolerede PP&#39;er nedsænkes i Optimal Cutting Temperature (OCT) medium, lynfrosset i flydende nitrogen og derefter gennemskåret med et cryomicrotome. Vævssnit (5 til 12 um) er lufttørret, fikseret med acetone eller methanol, og derefter underkastet immunolabeling.

Flowcytometrisk analyse af monodisperse suspensioner af den samlede PP-celler afslører en klar skelnen mellem gode og dårlige cellepræparater. I gode cellepræparater med over 80% levedygtighed, viser det store flertal af celler med høj forward scatter (FSC), en indikator for høj cellevolumen, og lav sidespredning (SSC), en indikator for lav celle granulering (figur 2A). I dette eksperiment har vi også bevidst fremstillet en &quot;dårlig cellepræparat&quot; ved inkubering PP-celler under isoleri...

Watch this Scientific Journal Video about Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches at JoVE.com

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Der er en stigende interesse for at forstå de immunologiske funktioner af specifikke subpopulationer af celler i Peyerske plaques (PP), de primære induktive steder af tarm-associerede lymfoide væv. Her skitserer vi parallelle protokoller til fremstilling PP encellede forberedelserne til flowcytometrisk analyse og PP kryosektioner for immunfarvning.

Isolering og immunfarvning Lymfocytter og dendritiske celler fra murine Peyerske plaks

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

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