D.E. and M.B.D. are members of a multidisciplinary research platform (MRP) of Ghent University, and the Ghent Researchers on Unfolded Proteins in Inflammatory Disease (GROUP-ID) consortium.

Bu çalışmada, yetişkin (6-8 haftalık) dişi C57BL / 6 fareleri kullanılmıştır. Fareler Gent Üniversitesi vivaryum kurallarına göre muhafaza ve yetiştirilen. Tüm hayvan prosedürleri Kurumsal Hayvan Bakım ve Etik Komitesi tarafından kabul edildi. Fare Dalak Mononükleer Hücreleri (MNC) hazırlanması 1. <ol><li> Servikal dislokasyon ile fare Kurban. </li><li> Geri aşağı diseksiyon gemide fare yerleştirin ve pimleri, arka ve ön ayaklarını düzeltmek. </li><li> % 70 ile deri yüzeyi sterilize (hacim / hacim) etanol / H2O </li><li> Toraks karın orta hat boyunca cildi kesin ve steril cerrahi aletler kullanılarak dalak maruz. </li><li> Çevresindeki yağ dokuları kırpma sonra, oda sıcaklığında 1 mi 1 x fosfat tamponlu tuzlu su (PBS) ihtiva eden bir 24 yuvalı plaka içine dalak yerleştirin. </li><li> Steril bir neşter kullanılarak dalak incelemek ve 50 ml&#39;lik küvet içine yerleştirilen 70 mikron naylon filtre içine parçaları aktarmake. </li><li> 2 ml şırınga pistonu kullanarak, yavaşça hücre süzgecinden dalak parçaları püre ve PBS 1x 5 ml ile yıkayın. </li><li> 15 ml&#39;lik bir tüpe Ficoll-Paque 3 ml ilave edilir ve 5 ml hücre süspansiyonu ile yoğunluk gradyan orta kaplar. </li><li> Fren RT&#39;de 20 dakika boyunca 400 x g&#39;de santrifüjleyin. </li><li> Taze bir 15 ml&#39;lik bir tüpe transfer interfaz 4 ° C &#39;de 10 dakika boyunca 300 x g&#39;de 10 ml soğuk 1 x PBS ve santrifüj ekleyin. </li><li> % 0.5 sığır serum albümini (BSA) ve 1 mM etilendiamintetraasetik asit (EDTA) (FACS tamponu) içeren PBS 1x 2 ml tekrar süspansiyon hücreleri. Aktarım, küçük hücre süspansiyonunun tüpe 10 ul,% 0.4 10 ul ile karıştırın </li></ol> 2. Zenginleştirme, Algılama ve Fare iNKT Hücreleri saflaştırılması <ol><li> Fare dalak CD5positive (CD5 +) lenfositlerin zenginleştirilmesi. <ol><li> (İsteğe bağlı) Leke 10 5 hücreleri FITC ile konjuge anti-CD5 (4 ul 1:30 seyreltme / 10 6 hücre)Karanlıkta 4 ° C&#39;da 30 dakika için, 200 ul FACS tamponu ve 500 tekrar süspansiyon FACS tamponu ul ile yıkayın. 1x PBS içinde 4 &#39;, 6-diamidino-2-fenilindol (DAPI) içindeki bir 20 uM çalışma çözeltisi hazırlayın, FACS tampon maddesi içinde ul bunun için 10 5 hücre 1 ekleyin ve 5 dakika boyunca oda sıcaklığında inkübe edilir. Akış sitometresinde 10 4 önceden zenginleştirilmiş hücreleri edinin. <ol><li> FCS-H ve FCS-W, hücre çiftleri ve agrega (Şekil 1A) dışlamak için FSC-W lo hücreleri üzerinde elektronik kapıyı kullanma. Ardından elektronik kapı dışarı DAPI + (ölü) FSC-W lo nüfus (Şekil 1B) içinde hücreleri ve elektronik boyut (Şekil 1C) tarafından lenfosit nüfus seçmek için SSC-A ve FCS-A kullanın. </li><li> Lenfosit kapısı içinde hücreleri kullanarak, CD5 karşı SSC-A arsa ve Şekil 1D gösterildiği gibi elektronik kapı CD5 + lenfositlerin. Akış sitometresinde öncesi zenginleştirilmiş numunenin kalanını edinin. </li&gt; </ol></li><li> FACS tampon 90 ul başına 10 7 hücreleri hücre sayımı ayarlayın ve 10 ul anti-CD5 mikroboncukları ekleyin. </li><li> 4 ml FACS soğuk FACS tampon tampon ve tekrar süspansiyon 500 ul ile bir kez yıkanır, 15 dakika boyunca 4 ° C&#39;de inkübe edin. Manyetik hücre ayrıştırma (MACS) kullanarak CD5 + lenfositlerin üreticinin önerilerine göre sütunlar için zenginleştirin. </li><li> FITC ile konjuge anti-CD5 opsiyonel) Leke 10 5 hücreleri (2.1.1 olarak 1:30 seyreltme / 10 6 hücre) ve DAPI 4 ul. Akış 10 4 zenginleştirilmiş lenfositlerin sitometresinde Edinme ve elektronik kapıları 2.1.1 oluşturulan doğrulayın. zenginleştirilmiş hücresel fraksiyonu CD5 + lenfositleri seçin. Akış sitometresinde 10 5 hücreleri Edinme ve Şekil 1E gösterildiği gibi zenginleştirilmiş fraksiyonu CD5 + lenfositlerin mevcut yüzdesini analiz - H. </li></ol></li><li> Algılama ve fare iNKT ce izolasyonuFACS ile lls. <ol><li> Anti-CD16 / CD32 içeren FACS tamponda yaklaşık 10 6 hücre başına 20 ul ve PE-konjuge αGalCer (1:50 seyreltme / 10 6 hücre 4 ul) içindeki bir konsantrasyonda zenginleştirilmiş CD5 + lenfositleri yeniden süspanse / CD1d tetramer (1 ug / 10 6 hücre). </li><li> Karanlıkta oda sıcaklığında 30 dakika süreyle inkübe edilir. </li><li> Fare antikorları (mAb &#39;ler), anti-CD3ε V500 (1:20 seyreltme 4 ul / 10 6 hücreleri), anti-CD 19 e450 (1:50 seyreltme 4 ul / 10 6 hücre) ve anti-CD8α e450 (4 ul seyreltik FACS tamponu içinde 1:50 seyreltme / 10 6 hücre), CD5 + lenfosit ekle ve karanlıkta 4 ° C&#39;de 30 dakika inkübe edilir. </li><li> 10 7 hücre / ml &#39;lik bir son konsantrasyonda 4 ml FACS tampon, 4 ° C&#39;de 10 dakika boyunca 300 x g&#39;de santrifüj ile tekrar süspansiyon 1 ml&#39;lik FACS tamponu ile hücrelerin yıkayın. </li><li> 30 mikron hücre süzgecinden hücreleri Filtre ve buz üzerinde hücreleri koyun. </li><li> Üretici tavsiyelerine göre 14,15 sıralama hücre sitometresinin akış kalibre. Not: hücre sıralama için bir akış sitometresinin Kalibrasyon eğitim gerektirir ve deneyimli bir operatör veya eğitimli bir teknisyen tarafından yapılabilir. </li><li> 10 6 hücre başına DAPI 20 uM çalışma çözeltisinin 10 ul ilave edin ve 5 dakika boyunca oda sıcaklığında inkübe edilir. Akış sitometresinde yaklaşık 10 4 zenginleştirilmiş CD5 + lenfositleri edinin. FSC-W lo hücreleri üzerinde elektronik kapı hücre çiftleri ve agrega (Şekil 2A) dışlamak için. Ardından elektronik kapı dışarı DAPI + CD8 + FSC-W lo nüfus (Şekil 2B) içinde CD19 + hücreler (döküm kanalı) ve 2.1.1.1 de anlatıldığı gibi elektronik lenfositler (Şekil 2C) seçmek için SSC-A ve FCS-A kullanın . <ol><li> CD3ε ve elektronik kapı αGalCer / CD1d tarafından arsa αGalCer / CD1d tetrameriŞekil 2D&#39;de gösterildiği gibi, tetramer + CD3ε + iNKT hücreleri. Zenginleştirilmiş hücresel fraksiyonunda mevcut yüzde αGalCer belirlemek için en fazla 5 10 x 2 den CD5 + zenginleştirilmiş lenfositleri Edinme / CD1d tetrameri + CD3ε + iNKT hücreleri. </li></ol></li><li> 2.2.7 oluşturulan elektronik kapıları kullanma. &#39;1.5&#39; bir akış hızında 70 mikron meme kullanılarak sitometresinde 70 psi akışı üzerinde, FACS sıralama αGalCer / CD1d tetrameri + CD3ε + iNKT hücreleri. 1 ml% 100 FCS ihtiva eden bir FACS tüpü içinde sıralanmış iNKT hücreler toplanır. Daha sonra 10 mi RMPI 1640 ortamında ve santrifüj 4 ° C &#39;de 10 dakika boyunca 300 x g&#39;de hücreler, FACS tüpü yanından kriteri iNKT hücreleri yıkayın. </li><li> Yeniden süspanse 1640 ortamı RPMI 1 ml iNKT hücreleri kriteri ve sıralanmış iNKT hücrelerinin saflığı analiz etmek üzere bir akış sitometresi üzerinde ul bunların 40 kazanır. Lenfosit - FSC-W lo DAPI seçins 2.2.7 tarif edildiği gibi. Elektronik ve kapı αGalCer / Şekil 2H gösterildiği gibi CD1d tetrameri + CD3ε + iNKT hücreleri - (G Şekil 2E). </li></ol></li></ol> 3. Kültür ve Fare iNKT Hücrelerinin Genişleme Gün 0 <ol><li> Kaplama oda sıcaklığında 1 saat boyunca hazne başına saflaştırılmış anti-CD3ε (3 ug / ml) ile, dibi düz 96 oyuklu plaka. 200 ul PBS ve bir kez 1 x 200 ul tam RPMI 1640 ortamında (% 10 hacim / hacim FCS, 100 U / ml penisilin-streptomisin, 5.5 uM β-merkaptoetanol) ile iki kez yıkanır. </li><li> 1.11 olarak% 0.4, bir hemositometre kullanılarak uygulanabilir kriteri iNKT hücreleri (aşama 2.2.8) sayısı., Tam RPMI 1640 ortamında 5 x 10 5 hücre / ml&#39;de, 4 ° C&#39;de 10 dakika boyunca 300 x g&#39;de santrifüje tabi tutulur ve tekrar süspansiyon Rekombinant fare IL-2 (10 ng / ml), rekombinant fare IL-12 (1 ng / ml) ve çözünür anti-fare CD28 (5 ug / ml) ihtiva eden. Plaka sAnti-CD3ε kaplı plakalar iyi 10 5 hücre / iNKT hücreleri orted ve 2 gün bir CO2 inkübatöründe 37 ° C&#39;de inkübe edin. </li></ol> Gün 2 <ol start="3"><li> 2 gün bir CO2 inkübatöründe 37 ° C&#39;de komple RPMI 1640 ortamı taze bir kaplanmamış düz dipli 96 oyuklu bir plaka ve kültür dinlenme koşulları altında, hücreler aktarın. </li></ol> 4. Gün <ol start="4"><li> Nazik pipetleme plaka hücreleri çıkarmak ve taze bir 15 ml tüp hücreleri aktarmak. Bir düz-10 5 hücre / göz, 4 ° C, rekombine IL-7 (5 ng / ml) ihtiva eden komple RPMI 1640 ortamında 5 x 10 5 hücre / ml&#39;de tekrar süspansiyon ve plaka 10 dakika boyunca 300 x g&#39;de santrifüjleyin Bir CO2 kuluçka makinesi içinde 37 ° C &#39;de 4 gün boyunca 96 plaka altlı. </li></ol> 8. Gün <ol start="5"><li> 300 x taze tüp hücreleri toplayın, santrifüjçözünür anti-CD28 (5 ug / ml) içeren tam RPMI 1640 ortamı içinde 5 x 10 5 hücre / ml&#39;de 10 4 ° C&#39;de dakika ve tekrar süspansiyon g. </li><li> Kaplama oyuk başına saflaştırılmış anti-CD3ε (3 ug / ml) ile, dibi düz 96 kuyucuklu bir plaka ve 3.1 gibi yıkayın. Levha 10 5 hücre / göz Anti-CD3ε kaplı plakalar ve gün 11 kadar bir CO2 inkübatör içinde 37 ° C&#39;de inkübe edin. </li></ol> Gün 11-18 <ol start="7"><li> Tekrarlayın 3.6 3.4 adımları tekrarlayın. </li></ol> Gün 19-20 <ol start="8"><li> Tam RPMI 1640 ortamında 5 x 10 5 hücre / ml&#39;de 300 x g&#39;de tekrar süspansiyon taze bir 15 ml tüp, santrifüj hücreleri aktarın. </li><li> Levha 10 5 hücre hücre / kuyuda bir düz tabanlı 96 oyuklu bir plaka ve izin hücreler bir deney içinde kullanılmadan önce bir CO2 inkübatör içinde 37 ° C&#39;de 2 gün süreyle dinlenmeye. </li></ol> 4. Sitokin üretimi CD1d Plat Kullanarak Fare iNKT Hücreleri tarafındane bağlı Deneyi <ol><li> Coat düz dipli 96 gözlü oyuk başına rekombinan sıçangil CD1d 100 ul (1 x PBS içinde 10 ug / ml) ile plaka ve 37 ° C&#39;de 1 saat boyunca inkübe edilir. </li><li> PBS 1x 200 ul ile dört kez yıkayın oyuk başına 1 x PBS (bloke çözeltisi) içinde% 2 FCS, 200 ul ekleyin ve 37 ° C&#39;de 2 saat inkübe edilir. </li><li> Blokaj çözeltisi kaldır, göz başına αGalCer 200 ul (5 ng / ml) ekleyin ve 16 st için 37 ° C &#39;de plaka inkübe edin. [ΑGalCer çözeltinin hazırlanması için, bir araç 96 mg / ml sakroz, 10 mg / ml sodyum deoksikolat ve 5 mg / ml Tween 20 ihtiva eden içinde çözülmüş αGalCer 55 ug 1085 μ 1x PBS ekleyin]. </li><li> Kuluçkadan sonra, αGalCer çözeltisi atılır ve 200 ul tam RPMI 1640 ortamı, ardından PBS 1x 200 ul de iki kez yıkayın. </li><li> Tam R santrifüj hücreler 300 x g&#39;de 10 dakika boyunca 4 ° C &#39;de ve tekrar süspansiyon, 21. günde, in vitro genişletilmiş iNKT hücreleri toplamak0.5 x 10 6 hücre / ml&#39;lik bir konsantrasyonda PMI 1640 ortamı. </li><li> Oyuk başına iNKT hücre yeniden süspansiyon 200 ul ilave edin ve bir CO2 inkübatör içinde 37 ° C&#39;de 72 saat boyunca inkübe edilir. </li><li> Üreticinin protokolüne göre, enzim bağlantılı immüno emici bir testle (ELISA) ile, ilgilenilen süpernatan ve ölçü sitokinler toplayın. </li></ol>

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The authors have no competing interests to disclose.

Geçerli protokol kritik adımlar izolasyon ve CD5 + lenfositlerin (Bölüm 1 ve 2) sonraki zenginleştirme, FACS sıralama (Bölüm 3) ve iNKT hücrelerinin ilk kaplama (Bölüm 4) yer alıyor. Bölüm 1&#39;de gerçekleştirilen adımların dikkatle ayrı bir hücresel interfaz santrifüj takip üretildiği şekilde yoğunluk gradyan ortamı üzerinden dalak hücresel süspansiyon katman unutmayın. Manyetik hücre ayırma (Bölüm 2) tarafından CD5 + lenfositlerin müteakip zenginleştirme leke...

Mürin değişmeyen doğal öldürücü T (iNKT) hücreleri CD1d-ifade kortikal timositleri 1,2 ile timus seçilen doğuştan T lenfositlerinin farklı bir nüfus vardır. iNKT hücreleri Vβ8, Vβ7 ya CD1d bağlamında endojen yanı sıra dış lipid antijenlerini tanıma yeteneğine sahip olan Vβ2 TCRler 3 ya ile eşleştirilmiş değişmeyen bir Vα14-Jα18 TCR zinciri içeren bir T hücre reseptörü (TCR) ifade etmektedir. Örneğin, kemirgen iNKT hücreleri tanıyan ve endojen yağ antijen olarak adlandırılan isoglobotrihexosylceramide (iGb3) 4, hem de α-galaktozilseramid (αGalCer) 5,6, deniz süngerlerinden izole edilen bir glikolipid ile aktive edilir. TCR bağımlı &lt;iNKT hücrelerinin aktivasyonu adaptif bağışıklık tepkilerinin teşvik hazırlanmasını, ve bunun sonucu olarak, iNKT hücreleri romatizmal hastalık 7 ve kanser dahil patolojilerin bir dizi iyileştirilmesi ya da geliştirme işlevsel olarak ilgili olduğu gösterilmiştirsup&gt; 8. Şu anda, sentetik iNKT hücre ligandlar immünopatolojik koşulları bir dizi düzenleme yeteneğine sahip olabilir umut vadeden yeni bir aşı yardımcı maddeleri oluşturmaktadır. Bu, daha önce iNKT hücreleri, ancak fare dokusundan izole aşağıdaki in vitro üretilebilir olduğu ortaya konmuştur; Bu çalışmaların bir çoğunda primer antijen sunan hücreler (APC&#39;ler) ve / veya hücre çizgileri 9 kullanımından yararlanmakta, Vα14 TCR transjenik (Tg) iNKT-hücresinden alınan hibridomadan 11,12 üretimi için farenin 10 veya timomalar. Bundan başka, farelerin çok sayıda, örneğin αGalCer yüklü CD1d dimerler ve uzun kültür zamanlarında gibi reaktiflerin yüksek hacimli bir yayınlanmış protokoller az etik ve ekonomik 9,13 çekici hale. Bu yazıda izolasyon ve fare dalak iNKT hücrelerin in vitro genişlemesi uyarlanmış bir yöntem tarif eder. Daha özel olarak ise, Protokol bir yöntem tarifFACS hücre sıralama için gerekli fareler, reaktifler ve süresini azaltır, ve in vitro sıralanan dalak iNKT hücrelerini genişletilmesi için optimize edilmiş bir yaklaşım önermektedir fare dalak iNKT hücrelerini zenginleştirmek için.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-2</td>
			<td>eBiosciences</td>
			<td>14-8021</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-12</td>
			<td>eBiosciences</td>
			<td>39-8122-65</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-7</td>
			<td>eBiosciences</td>
			<td>14-8071</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD3e (145-2C11)</td>
			<td>eBiosciences</td>
			<td>16-0032-86</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD28 (37.51)</td>
			<td>eBiosciences</td>
			<td>16-0281-85</td>
			<td></td>
		</tr>
		<tr>
			<td>e450-conjugated anti-mouse CD19 (eBio1D3)</td>
			<td>eBiosciences</td>
			<td>48-0193-82</td>
			<td></td>
		</tr>
		<tr>
			<td>e450-conjugated anti-mouse CD8&#945; (53-6.7)</td>
			<td>eBiosciences</td>
			<td>48-0081-82</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-conjugated anti-mouse CD5 (53-7.3)</td>
			<td>eBiosciences</td>
			<td>11-0051-81</td>
			<td></td>
		</tr>
		<tr>
			<td>V500-conjugated anti-mouse CD3&#949; (500A2)</td>
			<td>BD biosciences</td>
			<td>560771</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD5 (Ly-1) microbeads</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-049-301</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD16/32 (2.4G2)</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MS Columns</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue, 0.4% (wt/vol)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco</td>
			<td>12633-020</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Gibco</td>
			<td>10010-056</td>
			<td>[Ca2+/Mg2+ - free]</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Gibco</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-123</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4458-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>EDS-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque plus</td>
			<td>GE Healthcare</td>
			<td>71-7167-00 AF</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate F-bottom</td>
			<td>Greiner-bio one</td>
			<td>657-160</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Greiner-bio one</td>
			<td>665-180</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Greiner-bio one</td>
			<td>655-180</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml falcon tube</td>
			<td>Greiner-bio one</td>
			<td>188-271</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml falcon tube</td>
			<td>Greiner-bio one</td>
			<td>227-261</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m filter</td>
			<td>Greiner-bio one</td>
			<td>542-070</td>
			<td></td>
		</tr>
		<tr>
			<td>30 &#181;m filter</td>
			<td>Millipore</td>
			<td>SVGP01050</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS separator</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-jacketed CO2 incubator</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Kit</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSAria III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized method for isolating and expanding invariant natural killer t cells from mouse spleen

Hızla stimülasyon üzerine sitokinleri yeteneği değişmeyen doğal öldürücü T (iNKT) hücre soyunun işlevsel bir özelliğidir. iNKT hücreler bu bağışıklık yanıtlarını aktive ve direksiyon adaptif yeteneğine sahip doğuştan gelen bir T hücresi popülasyonu olarak karakterize edilir. Murin iNKT hücrelerin kültürü ve genişleme için geliştirilmiş tekniklerin geliştirilmesi, in vitro ve in vivo model sistemlerinde de iNKT hücre biyolojisi üzerinde çalışmayı kolaylaştırmaktadır. Burada, izolasyon ve murin splenik iNKT hücrelerinin genişlemesi için optimize edilmiş bir prosedürü açıklar. C57BL / 6 farelerinden alınan dalak çıkarılır, parçalara ayrılır ve süzülmüş ve elde edilen hücresel süspansiyon yoğunluk gradyan ortam üzerine yayıldı edilir. Santrifüj sonrasında, dalak mononükleer hücreler (MNC) toplanır ve CD5-pozitif (CD5 +) lenfositler manyetik boncuklar kullanılarak zenginleştirilmiştir. CD5 + fraksiyonu içinde iNKT hücreler daha sonra ^ ile boyanır5; CD1d tetramer GalCer yüklenmiş ve flöresanla aktive edilen hücre tasnif (FACS) ile saflaştırılmıştır. FACS murin yeniden birleştirici IL-7 varlığında genişletilmiş önce rekombinan sıçangil sitokin ve plaka bağlı T hücresi reseptörü (TCR) uyaran bir kombinasyonu kullanılarak iNKT hücreleri başlangıçta daha sonra in vitro olarak kültürlenir kriteri. Yaklaşık 10 8 iNKT hücreleri in vitro fonksiyonel deneyleri için ya da farelerde in vivo transfer deneyleri için de kullanılabilir ve bundan sonra kültür 18-20 gün içinde elde edilebilir Bu tekniği kullanarak.

Yoğunluk gradyanı kullanılarak dalak mononükleer hücrelerinin izolasyonu, yaklaşık 1 saat sürer ve kırmızı kan hücrelerinin (RBC) lize için gerekli reaktiflerin kullanımını ortadan kaldırır. Canlı hücreler yüksek verim alınır organın süzme sırasında üretilen bu yöntem ve tortuyu kullanılarak elde edilir. Tipik olarak, dalak lenfosit havuzu içinde iNKT hücrelerin frekansı, bu kullanılan hayvanların yaş, cinsiyet, sağlık durumuna bağlı olarak değişebilir, ancak toplam T lenfositle...

Watch this Scientific Journal Video about An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen at JoVE.com

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

Fare Dalak Değişmeyen Doğal Öldürücü T Hücreleri İzolasyon ve Yaygınlaştırılması için optimize Yöntemi

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

an-optimized-method-for-isolating-expanding-invariant-natural-killer

Research

JoVE Journal

Immunology and Infection

11.9K Görüntüleme.  Ghent University Hospital. Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents. 

Video: An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Fare T-hücrelerinin, T-hücre reseptörleri retroviral İletimi

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

İmmünoloji ve Enfeksiyon

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

Hazırlanması ve kullanımı, HIV-1 Enfekte İlköğretim CD4 + T-Hücreleri Hedef Hücreler olarak Natural Killer Hücre Sitotoksik Tahliller

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Kordon Kanı adlı Sitotoksik T lenfositler genişletilmesi olduğunu Hedef Sitomegalovirüs, Epstein-Barr Virus ve Adenovirüs

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Yapay Antijen Hücre (AAPC) Aracılı Aktivasyon ve Doğal Öldürücü T hücreleri genişletilmesi Presenting

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Etkin Killer yapay antijen sunan hücreler (KaAPC)<em&gt; İn Vitro</em&gt; İnsan Antijen-spesifik T hücrelerinin tükenmesi

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

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 ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

İnsan Tüm Kan Natural Killer Hücre Parçalayıcı Faaliyet Akışı Sitometrik Analizi

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Roman besleyici-Alerjik sistemi fare doğal katil hücreler Vitro seri üretim için

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Lenfositler fare küçük bağırsak bağışıklık sistemi yalıtma

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

Çok renkli akış sitometresi tabanlı Quantification mitokondri ve T hücrelerde organellerin

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...