Bu çalışma NIH PBS (R21 AI095484) tarafından desteklenmektedir. Biz CD1d tetramerin NHI tetramerdir çekirdek tesis ederiz.

1.. Araçların hazırlanması, Kültür Medya ve Reaktifler <ol><li> Araçlar: steril koşullar altında reaktifler hazırlamak ve bir laminar akış başlığı altında kullanmak. </li><li> Fetal sığır serumu% 5, bikarbonat, 2.1 g, 10 ml 100 x glutamat, HEPES, 10 mg, 1 mg sodyum piruvat ve 10 ml 100 x penisilin / streptomisin eklemek, RPMI doku kültür ortamı 1 L hazırlayın. Not: Önerilen doku kültür ortamı, RPMI, DMEM ile değiştirilebilir. </li><li> % 5 kolajenaz &quot;D&quot; çözeltisi (doku kültürü ortamında 9 ml kolajenaz &quot;D&quot; 5 ml) hazırlayın. </li><li> % 100,% 80 ve% 40: Üç farklı konsantrasyonlarda Percoll çözeltisi olun. Şu konsantrasyonlar 1 numune için hesaplanır. Oda sıcaklığında çözümler tutun. <ol><li> Percoll çözeltisi (stok çözeltiden) seyreltilmemiş Percoll 9 ml ekleyin; PBS 10x 1 ml ekleyin. </li><li> Percoll çözeltisi:; Percoll 8 ml% 100 eklemek PBS 1x 2 ml ekleyin. </li><li> Perkol çözüm:Percoll% 80 4 ml; 1x PBS içinde 4 ml. </li></ol></li><li> (FACS) tampon sıralama floresan-aktive edilmiş hücre için 1 L olun; 5 ml% 10 BSA (% 0.1 BSA), 1 ml% 10 sodyum azid (0.1%), 2 ml EDTA maddesi, 1 x PBS ile 1000 ml&#39;ye ekleyin. </li></ol> Böbrek sindirim 2. Hazırlanması <ol><li> , Gerekli kurumsal onayı edinilen standart yöntemlerle fareler kurban ve asıl bulbus olfaktoryusları alındı ​​ve mevcut yönetmelik ve hayvan bakım yönergeleri izleyerek olması. <ol><li> Pentobarbital (0.07 mg / kg) ile fare anestezisi. </li><li> Kansızlaştırma geçin. </li><li> Bir cerrahi masaya yatar pozisyonda fare yerleştirin. </li><li> % 70 etanol ile karın bölgesinde püskürtün. </li></ol></li><li> Petri kabına kollajenaz D çözeltisinin 10 ml ilave edilir. </li><li> Çıkarın ve her fareden her ikisi de böbrek açarlar. <ol><li> Için sternum karın derisi ve karın zarı aracılığıyla bir orta hat kesi yaparak üşüme boşluğu açınpubis. </li><li> Tarafına lateral olarak bağırsak hareket ile sol böbrek Açığa. </li><li> Dikkatlice forseps ile sol kapsülü ayrı. Not: Kapsül böbrek çevreleyen şeffaf bir katman olarak görünür ve kısmen perinefrik adipoz doku ile kaplı. </li><li> Bir cerrahi makas kullanarak pedikül damarlarını keserek sol böbrek çıkarın. </li><li> Sağ böbrek için aynı işlemi tekrarlayın. Not: sağ böbrek daha kranial ve orta hat (pedicels kısa olan) daha yakındır. </li></ol></li><li> Petri kabı içine böbrek yerleştirin. </li><li> Normal bir metal şekillendirme bıçağı kullanılarak küçük parçalar halinde böbrek doku (boyut olarak 1-2 mm) kesme ve (Şekil 1) 37 ° C kuluçka makinesi içinde 30-45 dakika boyunca kolajenaz çözeltisi içine parçaları yerleştirin. </li></ol> Böbrek sindirimden Böbrek Mononükleer Hücreleri (KMNC) 3 eklenmiştir. Hazırlanması <ol><li> Böbreğin, bir hücre süspansiyonu elde edilirmekanik olarak doku kültürü ortamı içinde 25 ml bir süzgeç (70 um) ve tekrar süspansiyon ile karıştırın doku bozarak sindirim. </li><li> Hücre süspansiyonu topak haline 4 ° C&#39;de 10 dakika boyunca 400 x g&#39;de santrifüjleyin. </li><li> Çok yavaş ve dikkatli bir şekilde% 40 Percoll çözeltisi 4 ml pelet yeniden askıya ve yavaşça bir transfer plastik bir pipet ile% 80 Percoll çözeltisi üzerine 4 ml kaplar. Bu açıkça bir saydam tabaka ile birbirinden ayrılmış iki aşamada neden olur. Bunun üzerine lipidler tekabül eden san bir tabaka olacaktır. </li><li> Fren kapalı modunda santrifüj ile oda sıcaklığında 30 dakika boyunca 1500 x g&#39;de santrifüjleyin. </li><li> Aspirasyon ile sarı ve kalın üst tabakası (yaklaşık 1-2 ml) sökün. </li><li> Bir transfer pipeti ile iki fazın arayüzünden hafif beyazımsı saydam tabaka toplayın. Çok dikkatli doku kültür ortamı ihtiva eden 2 ml, 15 ml konik bir tüp içine aktarmak ve bu süspansiyon daha sonra bir toplam hacmin orta bölgesinin 13 ml ekleyin15 mi. </li><li> 4 ° C&#39;de 10 dakika boyunca 400 x g&#39;de santrifüje </li><li> Ortamın 1 ml numune yeniden süspanse edin. </li><li> Bir menositometrede mavisi ile dışlama kullanarak KMNC sayısını saymak ve Böbrek başına ml başına KMNC numaraları gibi sonuçları ifade eder. </li><li> Bir kez yukarıdaki gibi yıkayın. </li></ol> KMNC 4. Hematopoetik İzolasyonu (CD45 +) Hücreler (Aşama I) <ol><li> Çalışan tampon 90 ul içine 10 7 hücreleri yeniden süspanse. </li><li> CD45 mikro-10 ul ilave edin ve 4 ° C&#39;de 15 dakika boyunca inkübe </li><li> Reaksiyonu durdurmak için çalışan tampon 1 ml ilave edilir. Çalışan tampon 500 ul, 4 ° C ve tekrar süspansiyon 10 dakika boyunca 400 x g&#39;de santrifüjleyin. </li><li> Mıknatıslarında yerleştirerek manyetik sütun hazırlayın ve çalışan tampon içinde 3 ml ile yıkayın. </li><li> Manyetik kolonundan hücre süspansiyonu geçmek ve sütundan geçer etiketlenmemiş hücreleri toplamak. </li><li> Runn 3 ml hücreler üç kez yıkayıntampon ing. Daha sonra çıkış maddesinin toplar. </li><li> Ayırıcı ve pipet sütunlar üzerine çalışan tampon 5 ml sütun çıkarın ve hemen etiketli kısmını dışarı floş. Bu fraksiyon, CD45 + hücreleri içerir. Hücreleri saymak. </li><li> Böbrekler, farklı T-hücreleri alt-mutlak sayısını hesaplamak için akış sitometrisi ile pozitif hücrelerin yüzdesi (Şekil 2) ile KMNC toplam sayısı çarpın. </li></ol> Negatif Seçim ile CD45 + Hazırlık DN T Cell 5. İzolasyonu (Aşama II) <ol><li> Aşağıdaki biyotinile edilmiş antikorların her biri 2.5 ul (0.5 mg / ml, 1.25 ug) ekleyin, anti-CD4, anti-CD8, anti-Fc reseptörü (CD16/32), anti-MHC class II (IA b), anti- Her 10 7 hematopoietik hücreler için CD1d PBS-57 tetramer. </li><li> Soğuk bir odada 30 dakika boyunca inkübe hücreleri. </li><li> 1 ml anti-Biotin, mikro-boncuklar ekleyin. </li><li> Soğuk bir odada 30 dakika boyunca inkübe edin. </li><li> T tüpü yerleştirinO mıknatıs ve tükenmesi geçin. </li><li> Bir hemasitometre kullanarak negatif fraksiyonunda DN T hücrelerinin toplam sayısını belirlemek için canlı hücreler sayılır. </li><li> Ilk kapısı CD45 +, ikinci kapısı TCRαβ +: Bu yolluk strateji izleyerek akış sitometri ile DN T hücrelerinin saflığını kontrol edin. CD1d + hücreleri hariç. CD8 + vs arsa CD4 + T hücrelerini DN (Şekil 3) tespit etmek. </li><li> Saflık yüzdesi ile belirlenen toplam sayısı çarpın. Not:% 90&#39;dan fazla saf DN T hücre popülasyonu içinde bu protokol kullanılması,. </li></ol>

<ol>
	<li>Zhang, Z. X., Yang, L., Young, K. J., DuTemple, B., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+previously+unknown+antigen-specific+regulatory+T+cell+and+its+mechanism+of+suppression.">Identification of a previously unknown antigen-specific regulatory T cell and its mechanism of suppression.</a> Nat Med. 6, 782-789 (2000).</li><li>Ford, M. S., Young, K. J., Zhang, Z., Ohashi, P. S., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+regulatory+function+of+lymphoproliferative+double+negative+T+cells+in+vitro+and+in+vivo.">The immune regulatory function of lymphoproliferative double negative T cells in vitro and in vivo.</a> J Exp Med. 196, 261-267 (2002).</li><li>Ford McIntyre, M. S., Young, K. J., Gao, J., Joe, B., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+in+vivo+trogocytosis+as+a+mechanism+of+double+negative+regulatory+T+cell-mediated+antigen-specific+suppression.">Cutting edge: in vivo trogocytosis as a mechanism of double negative regulatory T cell-mediated antigen-specific suppression.</a> J Immunol. 181, 2271-2275 (2008).</li><li>Young, K. J., Yang, L., Phillips, M. J., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-lymphocyte+infusion+induces+transplantation+tolerance+by+activating+systemic+and+graft-infiltrating+double-negative+regulatory+T+cells.">Donor-lymphocyte infusion induces transplantation tolerance by activating systemic and graft-infiltrating double-negative regulatory T cells.</a> Blood. 100, 3408-3414 (2002).</li><li>Chen, W., Ford, M. S., Young, K. J., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infusion+of+in+vitro-generated+DN+T+regulatory+cells+induces+permanent+cardiac+allograft+survival+in+mice.">Infusion of in vitro-generated DN T regulatory cells induces permanent cardiac allograft survival in mice.</a> Transplant Proc. 35, 2479-2480 (2003).</li><li>Young, K. J., DuTemple, B., Phillips, M. J., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+graft-versus-host+disease+by+double-negative+regulatory+T+cells.">Inhibition of graft-versus-host disease by double-negative regulatory T cells.</a> J Immunol. 171, 134-141 (2003).</li><li>Mohamood, A. S., 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=Gld+mutation+of+Fas+ligand+increases+the+frequency+and+up-regulates+cell+survival+genes+in+CD25+CD4++TR+cells.">Gld mutation of Fas ligand increases the frequency and up-regulates cell survival genes in CD25+CD4+ TR cells.</a> Int Immunol. 18, 1265-1277 (2006).</li><li>Ascon, D. 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W., 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=FcR+gamma+presence+in+TCR+complex+of+double-negative+T+cells+is+critical+for+their+regulatory+function.">FcR gamma presence in TCR complex of double-negative T cells is critical for their regulatory function.</a> J Immunol. 177, 2250-2257 (2006).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Bu tür otoimmün hastalıklar, kanser, graft tolerans ve akut böbrek hasarı (AKI) da dahil olmak üzere böbrek hastalıkları, birincil olarak farklı patolojik koşullarda karıştıktan çünkü DN T hücrelerinde artan bir ilgi vardır, 8, 13, glomerülonefrit. Bu nedenle, daha iyi bir DN T hücrelerinin patofizyolojik fonksiyonları ve karakterize etmek için bir ihtiyaç vardır. Ancak, şu anda CD4 ve CD8 T hücreleri ile karşılaştırıldığında, bu hücrelerin fonksiyonu içinde anlayışında...

A correction was made to: <a href="http://www.jove.com/video/51192/isolation-of-double-negative-t-cells-from-the-kidney">Isolation of Double Negative &#945;&#946; T Cells from the Kidney</a>.
There was an error with an author's name. The author's given name had a typo, this was corrected to:
Samatha Bandapelle
instead of:
Samantha Bandapelle

A correction was made to: Isolation of Double Negative &#945;&#946; T Cells from the Kidney.

Erratum: Isolation of Double Negative &#945;&#946; T Cells from the Kidney

Periferik αβTCR + CD3 + CD4 - CD8 - double-negatif (DN) T hücreleri fenotip ve işlevlerini 1-4 sahip çeşitli tiplerin ayrılır. DN T hücreleri, tam olarak anlaşılamamıştır, fakat çok daha farklı hastalık modellerinde 4-6 patofizyolojik bağışıklık tepkilerine dahil edilmektedir. DN T hücrelerinin artan çeşitli bağışıklık ve oto-bağışıklık tepkilerinin düzenlenmesi ve allotransplant tolerans modülasyonunda rol benzersiz bir bağışıklık hücresi bir türüdür. 4-6, 9 Bunlar dalak ve lenf düğümleri (periferal kan ve sekonder lemfoid organlarda nadirdir .) Ancak, normal böbrek ve bağırsak epitel 10-12 önemli sakinleri vardır. Çok az kararlı halde böbrekte işlevlerine yaklaşık 7 ve böbrek transpl ile bağlantılı akut böbrek hasarı (AKI) gibi patolojik koşullar altında bilinirkarınca. Çünkü her oyuncunun rolünü tanımlayan, alloresponses de dahil olmak üzere bağışıklık tepkilerini düzenleyen farklı bağışıklık hücrelerinin karmaşık rolleri alloresponses anlamak ve yeni terapötik tasarımı önemlidir. Ve fizyolojik koşullar altında, farklı hastalık, böbrekte bulunan DN T hücrelerinin önemli sayıda göz önüne alındığında, DN T hücreleri transplant alıcılarında kritik farelerde ve insanlarda bağışıklık ve oto-bağışıklık tepkileri düzenlenmesinde bir rol oynayabilir ve alloresponses muhtemeldir. Hala dağınık delil olsa Biriken hem patojenik ve bağışıklık fonksiyonları DN T hücrelerini implicates ama neden ve nasıl belli bir zararlı ya da baskılayıcı fonksiyonunu sergilemek ve çevre onları nasıl etkilediğini de anlaşılamamıştır. Nedeniyle böbrekte düşük bolluğu nedeniyle, izolasyon geliştirilmiş yöntemler gerekmektedir. T DN T hücrelerinin izole edilmesi için standart bir protokol bulunmamaktadıro non-lenfoid dokular. Bizim protokol böbrekten DN T hücrelerinin izole edilmesi için yeni bir yöntem tarif eder; Bununla birlikte, yöntem, aynı zamanda, çeşitli lenfoid olmayan dokular için kullanılabilir.

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			<td>Or equivalent equipment&#160;</td>
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			<td height="21" style="height:21px;width:439px;">Analytical flow cytometer</td>
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			<td height="20" style="height:20px;width:439px;">RPMI 1640</td>
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			<td height="20" style="height:20px;width:439px;">Buffer phosphate buffered saline (PBS)</td>
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			<td height="21" style="height:21px;width:439px;">PBS 10X</td>
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			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">CD45+ microbeads</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">6.78E-51</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Biotin microbeads&#160;</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">130-090-485</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD45 (clone: 30-F11)&#160; PerCP&#160;</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td style="width:276px;">G1397</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD45 (clone: 30-F11)&#160; APC-Cy7</td>
			<td style="width:257px;">Biolegend&#160;&#160;</td>
			<td style="width:276px;">103116</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-TCR Pacific Blue&#160; (ab-chain, clone: H57-597)</td>
			<td style="width:257px;">Invitrogen</td>
			<td style="width:276px;">HM3628</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD4 PE</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td>12-0043</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD8 FITC</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td>8011-0087</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD4 biotinylated (GK1.5)</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553728</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD8 biotinylated&#160; (clone 53-6.7)</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553029</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Fc receptor (CD16/32) biotinylated</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553143</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-MHC class II (I-A d) biotinylated</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553609</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD1d PBS-57 tetramer</td>
			<td style="width:257px;">NHI tetramer&#160;</td>
			<td style="width:276px;">lote # 15621-PBS57</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:439px;">AutoMACS Running Buffer-&#160; MACS Separation Buffer&#160;</td>
			<td style="width:257px;">MIlteny I Biotec</td>
			<td style="width:276px;">130-091-221</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">C57BL/6J mice</td>
			<td>Jackson Labs</td>
			<td style="width:276px;">000664</td>
			<td>Kidneys - Lymph Nodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Petri dish&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:276px;">356517</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">70mc filter&#160;</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Plunger&#160;</td>
			<td style="width:257px;"></td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GeneMate Tubes 50 ml</td>
			<td>Bioexpress</td>
			<td>C-3394-3</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">GeneMate Tubes 15 ml</td>
			<td>Bioexpress</td>
			<td style="width:276px;">C-3394-1</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Petri Dish&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:276px;">S33580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Plate 24 wells&#160;</td>
			<td style="width:257px;">BD Biosciences</td>
			<td style="width:276px;">354723</td>
			<td style="width:200px;"></td>
		</tr>
	</tbody>
</table>

isolation of double negative &#945;&#946; t cells from the kidney

Lenfoid olmayan dokulardan DN T hücrelerinin izole edilmesi için standart bir protokol, çeşitli immün yanıtların onların daha çok rapor katılımı rağmen bulunmamaktadır. DN T hücreleri Allotransplantlar 1-6 bağışıklık ve oto-bağışıklık yanıtlarını ve tolerans düzenlenmesinde implike edilmiştir özel bir immün hücre türüdür. DN T-hücreleri, periferik kan ve sekonder lemfoid organlarda (dalak ve lenf düğümleri) nadir, ancak, fakat normal böbrekte önemli sakinleri vardır. Çok az nedeniyle çevrede kendi azlığı onların patofizyolojik işlevi 7 hakkında bilinmektedir. Biz son zamanlarda sürekli halde ve iskemi reperfüzyon yaralanması esnasında böbrek 8&#39;de bu nüfusun kapsamlı bir fenotipik ve fonksiyonel analiz nitelendirdi. DN T hücresi işlevinin analizi büyük ölçüde böbrek kendi izolasyon için bir protokol geliştirilerek artırılacaktır. Burada, biz isolatio sağlayan bir roman protokol açıklarMurin böbrekten oldukça saf ab CD4 + CD8 + T hücreleri ve T hücre DN n. Kısaca, biz kollajenaz kullanılarak böbrek doku sindirmek ve yoğunluk gradyanı ile böbrek mononükleer hücreleri (KMNC) izole. Bu KMNC den% 70 &#39;den% 3 hematopoietik T hücreleri zenginleştirmek için iki aşama takip eder. İlk adım, CD45 + izolasyon kiti kullanılarak hematopoietik hücrelerin pozitif seçim oluşur. İkinci aşamada, DN T hücreleri negatif CD4, CD8 ve MHC sınıf II monoklonal antikorlar ve CD1d α-GalCer tetramer kullanılarak olmayan arzu edilen hücreler ayrılmasıyla izole edilmektedir. Bu strateji,% 90&#39;dan fazla saf DN T hücre popülasyonu yol açar. , FACS analizi ile, ardından yukarıda belirtilen antikorlar ile lekeleme yüzey saflığını teyit etmek için kullanılır.

Yabani tip C57BL / 6 (B6), böbrek böbrek başına yaklaşık 1.5-2.1 x 10 6 mononükleer hücreleri içerir. Yaklaşık olarak en fazla% 10 CD45 + hematopoietik hücrelerdir. % 5 kolajenaz ile sindirme ile ve ardından, Şekil 1&#39;de gösterildiği gibi böbrek tek çekirdekli hücreleri (KMNC) hazırlanması için, böbrekler küçük parçalar halinde kesilir. Bu KMNC tabakası (Şekil 2, sol panel) toplamak için, bir yoğunluk g...

Watch this Scientific Journal Video about Isolation of Double Negative Î±Î² T Cells from the Kidney at JoVE.com

Isolation of Double Negative &amp;#945;&amp;#946; T Cells from the Kidney

DNabT hücreleri periferal T-hücreleri arasında nadirdir; Bununla birlikte, belli olmayan lenfoid dokuda bol miktarda bulunmaktadır. Non-lenfoid doku DN T hücrelerini izole Zorluk patofizyolojik önemini kabul kapasitelerine rağmen fonksiyonel analizini engellemektedir. Bu murin böbreğinden yüksek derecede arıtılmış DN T hücrelerinin izole edilmesi için yeni bir protokol açıklar.

Böbrek gelen Çift Negatif αβ T Hücreleri İzolasyon

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

isolation-of-double-negative-t-cells-from-the-kidney

Research

JoVE Journal

Immunology and Infection

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

12.1K Görüntüleme.  Johns Hopkins University School of Medicine.  DNabT hücreleri periferal T-hücreleri arasında nadirdir; Bununla birlikte, belli olmayan lenfoid dokuda bol miktarda bulunmaktadır. Non-lenfoid doku DN T hücrelerini izole Zorluk patofizyolojik önemini kabul kapasitelerine rağmen fonksiyonel analizini engellemektedir. Bu murin böbreğinden yüksek derecede arıtılmış DN T hücrelerinin izole edilmesi için yeni bir protokol açıklar. 

Video: Isolation of Double Negative &#945;&#946; T Cells from the Kidney

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

İlköğretim İnsan naif ve Bellek T Hücreleri gelen İsteyerek Düzenleyici T Hücreleri Üretimi

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

İmmünoloji ve Enfeksiyon

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

İzolasyon ve naif CD4 T Lenfositler Th17 Farklılaşma

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

T hücrelerinin türetilmesi<em&gt; İn Vitro</em&gt; Fare Embriyonik Kök Hücreleri

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Periferik Kan İnsan alloantijen-özgü T hücrelerinin üretilmesi

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Fare Naif CD4<sup&gt; +</sup&gt; T Hücre İzolasyonu ve<em&gt; In vitro</em&gt; T hücre alt içine Farklılaşma

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T hücreleri dendritik hücreler gelen Transinfection tarafından Bakteriler Yakalama

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

İnsan Palatine Bademcikler B ve T Lenfosit için Verimli İzolasyon Protokolü

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

İnsan Endoservikal Gamma Delta T Hücreleri İzolasyon ve Akım Sitometrisi Analizi

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

Periferik kan ve lenf dokusu birincil insan T hücre sinaptik arabirimi değerlendirilmesi

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Çift iplikçikli RNA ve desen tanıma reseptörleri negatif anlamda RNA virüsü enfeksiyonu confocal görüntüleme

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...