We are grateful to Dr. Jos&#233; Luis Daniotti for comments. We thank the assistance to Dr. J. Arturo Pimentel and the Laboratorio Nacional de Microscop&#236;a Avanzada-UNAM for acquisition of confocal images. I.M.-D. and T.M.V.C. were supported by grants 157634 and 253596 from Consejo Nacional de Ciencia y Tecnolog&#236;a (CONACYT), Sociedad Latinoamericana de Glicobiolog&#236;a, A.C. T.M.V.-C. is recipient of a scholarship (245192) from CONACYT. We also thank the support of the Red Tem&#225;tica Glicociencia en Salud - CONACYT (253596).

Sağlıklı erkek vericilerden alınan periferik kan aydınlatılmış onam ve Centro de INVESTIGACION en DINAMICA Celular- Universidad Autonoma del Estado de Morelos Biyoetik Komitesi onayı ile elde edilmiştir. 1. İzolasyon ve insan naif CD4 + T hücrelerinin aktivasyonu <ol><li> bilgilendirilmiş rıza ile Sağlıklı insan donörlerden elde edilen periferal kan 2 mL kan ve steril PBS-EDTA, 2 ml seyreltin (1x Fosfat tamponlu tuz (PBS), 2 mM EDTA, pH 7.4) eklenmiştir. Daha önce tarif edildiği gibi 8 1.077 yoğunlukta sukroz çözeltisi 3 ml üzerine yavaşça seyreltilir kan ekleyin. Not: yoğunluğundaki farklar nedeniyle santrifüj sırasında Diferansiyel geçiş eritrositler tamamen çöktürmek için neden olur ve daha az yoğun bir mononükleer hücre tabakası, yukarıda oluşturacaktır. Periferik kan 2 mi cevap verecek sonra yaklaşık 0.5 x 10 6 naif CD4 + T hücrelerinin hale getirecekadımlar. </li><li> Resim arası 21 ° C (30 mm ayarlanmış bir açıyla yuvarlak tabanlı borular için kullanımı rotor) 250 x g&#39;de santrifüjleyin 30 dakika periferal kan tek-çekirdekli hücreleri (PBMC) gradyanı üretir. Santrifüj işleminden sonra PBMC&#39;ler açık beyaz bir halka olarak gözlemlenebilir. bir pipet PBMC&#39;lerin toplamak ve yıkama için steril bir tüpe transfer. </li><li> 10 dakika boyunca 250 x g&#39;de PBS-EDTA çözeltisi ile üç yıkamadan sonra, saf CD4 + T hücrelerinin saflaştırılması geçin. Sıralama metodları veya saflaştırma kitleri çeşitli tipleri bu amaçla kullanılabilir. Tercihen, saflaştırma için&gt; 1 x 10 7 PBMC&#39;ler kullanın. <ol><li> Manyetik boncuklar ile negatif seleksiyon tarafından naif CD4 + T hücreleri arındırmak. Anti CD45RO, CD8, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD56, CD123, anti-TCRγ / δ, anti-HLA-DR ve CD235a (glikoforin A) antikorları ile negatif seçim gerçekleştirin. f ile CD4 + CD45RA + hücrelerinin belirlenmesi ile saflık yüzdesi değerlendirilmesiDüşük sitometri. Not: İsteğe bağlı olarak, bir gece boyunca 37 ° C&#39;de ve% 5 monosit yapışmasını teşvik etmek ve naif CD4 + T hücresi saflaştırılmadan devam etmek için takviye edilen ortam, 10 ml, 1 x 10 6 hücre / ml&#39;lik bir yoğunluğa sahip CO 2 PBMC&#39;lerin inkübasyonundan geçin. Periferik kandan naif CD4 + T hücrelerinin etkili bir şekilde geri ölçüde saflaştırma sistemine bağlıdır. </li></ol></li><li> 5 ug / ml anti CD3 monoklonal antikor ile PBS göz başına 250 ul ilave edilerek 24-iyi hücre kültür plakaları hazırlayın ve 37 ° C&#39;de en az 2 saat süreyle inkübe edin. Not: 96 kuyucuklu levhalar kullanılabilir naif CD4 + T hücrelerinden daha az sayıda kullanıldığında (örneğin, 2 x 10 -1 x 10 5 4). </li><li> 24 de ya da 96 gözlü levhalar anti CD3 antikoru seyreltme çıkarın ve iki kez steril 1 x PBS kullanarak plakaları yıkayın. </li><li> + Saflık&gt;% 95 ile naif CD4 + T hücreleri (CD4 sulandırmakFetal sığır serumu% 3 ile takviye edilmiş, gelişmiş RPMI 1640 ortamı içinde 1 x 10 6 hücre / ml&#39;lik bir yoğunluğa CD45RA +) (veya RPMI serumu% 10), 2 mM glutamin ve antibiyotikler (1 U / ml penisilin, 1 ile desteklenmiş ug / ml streptomisin). Not: Gangliozidleri yerelleştirme ölçülerek gerekiyorsa aşağıda tarif edildiği gibi, seyreltme ve uyarılması için aynı protokolü uygulayın. </li><li> Anti CD3 kaplı oyuklara 24 plaka olmayan kaplı oyuklara (kontrol hücreleri) hücre seyreltme 500 ul ekle. Seçenek olarak ise, 96 gözlü bir levhanın gözleri hücre seyreltme 100 ul ilave edin. </li><li> Anti CD28 antikoru 1 ug / ml ekleyerek, anti CD3 kaplı kuyucuklara naif CD4 + T hücrelerinin uyarılması koşulları doldurun. </li><li> Anti CD28 antikorlarının ilavesi olmadan olmayan kaplanmış kuyucuklara kontrol olarak dinlenme CD4 + T hücreleri tutun. 37 ° &#39;lik standart koşullar altında 72 saat için 0 dinlenme ve aktive CD4 + T hücresi inkübeC ve% 5 CO2. </li><li> 37 ° C&#39;de inkübe naif CD4 + T hücreleri olarak CD69 erken aktivasyon işaretleme (16 saat sonra aktivasyon) ya da CD25 geç aktivasyon işaretleme (48 saat sonra etkinleştirme) ekspresyonu akış sitometresi ile değerlendirilmesi ve aktive edilmiş uygun bir aktivasyon kontrol etmek için ve% 5 yokluğunda veya anti CD3 / CD28 antikorları 9,10 mevcudiyetinde CO2. </li></ol> 2. İstirahat ve bağlılık Olmadan Naif CD4 + T hücreleri <ol><li> en az 15 dakika süre ile otoklavlama ve UV ışığı maruz bırakılarak 12 mm yuvarlak lamelleri sterilize edin. </li><li> Steril 24 oyuklu hücre kültür plakalarında yer lamelleri. Not: 1&#39;den daha x 10 5 hücre olan kültürler için, lamel hücre kaybını önlemek üzere uyarlanmış küçük kuyu slayt odaları kullanılması tercih edilir. </li><li> Kat lamelleri poli-L-lisin, 200 ul (veya sürgülü bölmesi) (MA 150-300 kDa),% 0.1 (ağırlık / hacim) çözeltisi ve r, 5 dakika boyunca inkübeoom sıcaklığında (RT), oda sıcaklığında en az 1 saat boyunca çıkarın ve kurutulur. </li><li> Dikkatli bir şekilde karıştırın ve 24 oyuklu plakalar istirahat ve aktif naif CD4 + T hücreleri toplamak. Hücre sayımı ve gerekirse poli-L-lizin kaplı kapak 1 x 10 5 hücre transferi taze desteklenen ortamda ile ayarlayın. 37 o C&#39;de 6 saat minimum ve% 5 CO 2 hücreleri inkübe. </li></ol> 3. Naif CD4 + T Hücreleri Toplama ve İstirahat Fiksasyon Olmadan <ol><li> Dikkatle kültürlü dinlenme ve aktif naif CD4 + T hücrelerinde bulunan ortamı çıkarın. </li><li> RT steril PBS&#39;nin 500 ul yavaş yavaş yapın. Not: kuyu çözümleri dikkatli toplama ve çıkarma lamel hücreleri çıkmasını engeller. </li><li> PBS atın ve iyice kültür ortamı çıkarmak için başka bir 500 ul PBS ekleyin. </li><li> 500 ul süzüldü% 4 paraformaldehid ilave (filt hücreleri saptamakrasyon mikroskopi analizi ile müdahale) ve (tercih edilen) veya gece boyunca 4 ° C&#39;de oda sıcaklığında 30 dakika inkübe edilir olabilir mikro kaldırır. Dikkat: Paraformaldehit toksik ve uçucu bir bileşiktir. Bir insan kanserojen olduğu bilinmektedir. Uygun bir güvenlik protokolü ile dikkatlice kullanın. </li><li> Fiksatif çıkarın ve oda sıcaklığında PBS ile en az iki kez yıkayın. </li><li> bir sonraki adıma devam veya boyama kadar birkaç gün 4 ° C&#39;de 500 ul PBS ile plaka kuyuları korumak. Not: Bu işlem sırasında Sterilite mikroorganizma büyümesini önlemeye yardımcı olur. </li></ol> 4. Boyama, Konfokal Mikroskopi Montaj ve Görselleştirme <ol><li> kuyulardan PBS kaldırmak. Soğuk bloke edici tampon 500 ul ekle (1X PBS,% 0.5 Standart kalite sığır serum albümini,% 1 fetal inek serumu, pH 7.4) eklenmiştir. Buz üzerinde 20 dakika süreyle inkübe edin. </li><li> dikkatlice engelleme tamponu çıkarın ve primer antikorlar ekleyin (anti GD3 klon R24, GD2 klon 14G2a),PE-konjuge anti CD25 ve APC-konjuge anti TCR ve 2.5 ug / ml bloklama tamponu içerisinde seyreltilmiş kontrol izotipleri. </li><li> Buz üzerinde 2 saat veya gece boyunca 4 ° C inkübe edin. Not: Yavaş yörünge ajitasyon tercih edilir. </li><li> dikkatlice antikor çıkarın ve PBS yavaşça 500 ul ekleyin. İki kez tekrarlayın. </li><li> İkincil antikorlar 0.1 ug / ml bloklama tamponu içinde seyreltilmiş (R24, anti GD3, Alexa Fluor 14G2a karşı GD2 488-konjuge veya Alexa Fluor 647-bağlı anti IgG2a için FITC ile birleşik anti-IgG3) ekleyin. </li><li> çalkalamadan karanlıkta buz üzerinde 1 saat süreyle inkübe edin. </li><li> 4.4&#39;de tarif edildiği gibi) PBS içinde üç kez yıkanır. Örneklerin dehidratasyon önlemek için yeterli PBS ile tutun. </li><li> 0.1 ug / ml, PBS içinde seyreltilmiş Hoechst 333.258 leke ekleyin. </li><li> Oda sıcaklığında 15 dakika inkübe ve çıkarın. PBS içinde üç kez yıkanır. </li><li> bir kağıt havlu üzerinde slaytlar yerleştirin ve montaj çözeltisi (PBS içinde% 50 gliserol) veya ticari monte çözeltisinin 20 uls solma ve numunelerden söndürme önlemek için. </li><li> Dikkatlice ince cımbız ile lamel pick up ve montaj çözümü üzerine yerleştirin. Hücreler bağlandıkları lamel yan çözeltisi ile temas içinde olduğundan emin olun. oje ile lamelleri Seal ve floresan veya konfokal mikroskopi kullanılarak analiz. </li></ol> RNA izolasyonu 5. <ol><li> Anti CD3 antikoru (5 ug / ml) kaplı 96 oyuklu plakalar hazırlamak. Uyaran tamamlamak için 4 x 10 4 naif CD4 + T hücre / oyuk anti CD28 antikoru (1 ug / ml) ile takviyeli kültür ortamında 100 ul inkübe edin. 0 ila 72 saat boyunca CO2 37 ° C&#39;de inkübe edilir ve% 5. </li><li> Farklı sonrası aktivasyon süreleri sonra bir mikrosantrifüj tüp içine hücreleri yerleştirin. </li><li> Oda sıcaklığında 5 dakika boyunca 250 x g&#39;de 500 ul PBS ve santrifüj ekleyin. </li><li> Süpernatantı ve 1 ml Trizol reaktifi ekleyin. </li><li> Oda sıcaklığında 5 dakika boyunca inkübe edin ve en örnek devam -8076, en az 24 saat bekletilmiştir. gerektiğinde RNA izolasyonu ile devam edin. Not: en az 24 saat boyunca -80 ° C&#39;de örnek bırakılması RNA verimini geliştirir. Ayrıca, el eldiven kullanımı RNases RNA bozulmasını önlemek için gereklidir. </li><li> Bir su banyosu içinde 2 dakika için 37 ° C&#39;de inkübe edilerek örnek Defrost. </li><li> 200 ul kloroform ilave edin ve 30 sn (vorteks RNA agresif karışmasını önlemek için) boyunca elle kuvvetli bir şekilde çalkalanır. Oda sıcaklığında 5 dakika inkübe edin. </li><li> 4 ° C&#39;de 12,000 x g&#39;de santrifüjleyin 15 dakika. </li><li> Yeni bir mikrosantrifüj tüpü içine, sulu faz yeniden elde. </li><li> 500 ul izopropanol ekleyin ve tüpü üç kez ters çevirin. </li><li> 4 ° C&#39;de 12,000 x g&#39;de 10 dakika süre ile 10 dakika oda sıcaklığında ve santrifüj inkübe edin. </li><li> tersine çevirerek süpernatant atın. </li><li> 4 ° C&#39;de 12,000 x g&#39;de buz soğukluğundaki% 75 etanol ve santrifüj 10 dakika için 1 ml ilave edilir. </li><li> Tamamen süpernatantı atmak ve açık tüpün kapağını bırakarak RNA pelet kurulayın. Değile: Bu noktada pelet açıkça görülebilir değildir. Yine de devam edin. </li><li> moleküler sınıf eden 20 ul pelletini. Nanodrop kullanılarak 260 nm ve 280 nm absorbans ölçümü ile konsantrasyon ve nükleik asitlerin saflık hesaplayın. </li><li> alışılmış herhangi bir ters transkriptaz kiti kullanılarak ve imalatçının protokolü izlenerek cDNA sentezi için dikkatli bir şekilde devam ediniz. Not: Yaklaşık 0.5-2 ug RNA, 4 x 10 4 naif CD4 + T hücrelerinden elde edilir. cDNA, RNA, 100 ng sentezlenmiş ve düşük RNA giriş kitleri gerek kalmadan, gerçek zamanlı PCR reaksiyonlarında kullanılabilir. </li></ol>

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The authors have nothing to disclose.

Açıklanan protokol, CD4 + T hücreleri ya da küçük bir hücre sayısı başlayarak, diğer bağışıklık hücreleri (örneğin, ekspresyonu analiz edilmiştir, Şekil 5) hücre süspansiyonlarında gangliosidler veya protein lokalize etmek için de kullanılabilir. Çünkü T hücreleri ve yapışmaz özellikleri küçük boyutu, kötü bilgi veya düşük kalitede floresan mikroskobik görüntüleri sonuçların elde edilmesi hücreleri doğru yapıştırılır de?...

CD4 + T hücreleri, antijen sağlayan hücreler 1 tarafından aktive edildikten sonra, bunların etki edici fonksiyonları ile bağışıklık yanıtını yönlendirirler. etkinleştirme sırasında modüle hücresel mekanizmaların çalışma bağışıklık fonksiyonunun temel bir süreci içine fikir verir. Kan çevre 2 hücrelerinin çok küçük bir popülasyonu temsil Ancak, saf CD4 + T hücrelerinin çalışması zor olabilir. Floresan mikroskobu aracılığıyla çeşitli raporlar CD4 + T hücresi aktivasyonu yer alan farklı moleküller plazma membranında 3 ile ilişkili temel protein lokalizasyonu inceledik. Gangliositler sialik asit içeren glikosfingolipidler ve onlar yaygın böyle bağışıklık hücreleri gibi bol, diğer hücreler sinir hücreleri incelenmiştir rağmen, aynı zamanda biyolojik ilgili fonksiyonları 4,5 ile gangliositler ifade eder. Biz daha önce tha bildirdiST8Sia 1 (GD3 sentaz) ve GM2 / GD2 sentaz sıyalıltransferaz α2,8 bir düzenlenmesi görülür, insan naif CD4 + T hücrelerinin aktivasyonu sırasında bulunan t, yani GD2 önemli yüzey neoexpression ve GD3 gangliozide 6 düzenlenişini uyarmaktadır. bağışıklık hücrelerinde GD3, GD2 ve gangliosidler daha başka çalışma, bağışıklık fonksiyonunun bir protein bazlı kısmi görünümü güzelleştirmek için gereklidir. Genel olarak, gangliosid ifade çalışması, ince tabaka kromatografisi (TLC), 7 gibi teknikler dayanan, ancak bu teknik, biyolojik analizi sınırlayıcı plazma membranı veya hücre içi bölümlerinde gangliosidler uzamsal lokalizasyonu izin vermez. Bu çalışmada, anti-CD3 / anti-CD28 aktivasyonundan sonra, insan naif CD4 + T hücreleri ve PBMC&#39;de GD3 ve GD2 antikor aracılı tanımlama ve lokalizasyonu için bir protokol açıklamaktadır. Bu protokolü i ilelenfositlerin küçük boyutlu (9 mm) dikkate alınarak, yüksek kaliteli görüntüler 6 satın alınmasından sonra, süspansiyon hücrelerin düşük sayıda gangliosidler geni ve moleküler ifadesini analiz etmek de mümkündür.

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		<tr height="21">
			<td height="21" style="height:21px;width:481px;">Advanced RPMI 1640&#160;</td>
			<td style="width:241px;">Thermo Fisher Scientific</td>
			<td style="width:137px;">12633-012</td>
			<td style="width:193px;">Suplemented with 3% FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human CD3 antibody</td>
			<td>eBioscience</td>
			<td>16-0037-81</td>
			<td>OKT3 clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human CD28 antibody</td>
			<td>ebioscience</td>
			<td>16-0288-81</td>
			<td>CD28.6 clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human GD3&#160; antibody</td>
			<td>Abcam</td>
			<td>ab11779</td>
			<td>R24 clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human GD2 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-53831</td>
			<td>14G2a clone</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:481px;">FITC-conjugated anti-mouse IgG3 
			antibody</td>
			<td>Abcam</td>
			<td>ab97259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 conjugated antimouse</td>
			<td>Thermo Fisher Scieni&#161;tific</td>
			<td>A-21131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG2a antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 647 conjugated antimouse</td>
			<td>Thermo Fisher Scieni&#161;tific</td>
			<td>A-21241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG2a antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 333258</td>
			<td>Sigma-Aldrich</td>
			<td>861405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Ficoll Paque-Plus</td>
			<td>GE</td>
			<td>17-1440-02&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Naive CD4+ T Cell Isolation Kit II, human</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-094-131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACSAria II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Sorting&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Lab-tek chamber slide system</td>
			<td>Sigma-Aldrich</td>
			<td>C7182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus FV 1000 Laser Confocal Microscope (Olympus)&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td>Upright BX61WI and IX81&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forward sequence primer GD3 synthase</td>
			<td>5&#180;-GAGCGTTCAGGAAACAAATGG- 3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reverse sequence primer GD3 synthase</td>
			<td>5&#180;-CCTGTGGGAAGAGAGAGTAAG-3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forward sequence primer GM2/GD2 synthase</td>
			<td>5&#180;-CAACACAGCAGACACAGTCC-3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reverse sequence primer GM2/GD2 synthase</td>
			<td>5&#180;-GTGGCAATCGTGACTAGAGC-3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
	</tbody>
</table>

preparation of cd4+ t cells for analysis of gd3 and gd2 ganglioside membrane expression by microscopy

Süspansiyonda saf CD4 + T hücrelerinin aktivasyonu için, burada tarif edilen yöntem ve konfokal mikroskopi analizi için lamelleri bunların yapışma deneyleri profil komplemanın sentezleme akış sitometrisi gibi, CD4 + T hücresi aktivasyonunda rol gangliosidler uzamsal lokalizasyonu ve görselleştirme izin batı blotting ya da gerçek-zamanlı PCR. akış sitometrisi ve mikroskopi yoluyla hücresel lokalizasyon yoluyla gangliosid ifadesinin ölçümü, yüksek yakınlık ve özgüllük ile anti-gangliosid antikorların kullanılması ile elde edilebilir. Bununla birlikte, süspansiyon içinde hücrelerin yeterli bir kullanım floresan veya konfokal mikroskopi edinimi için gerekli olan yapışma teşvik etmek için kültür plakaları muamele edilmesini içerir. Bu çalışmada, saf CD4 + T hücresi aktivasyonu sırasında GD3 ve GD2 gangliozid ekspresyonunu ve TCR ile ko saptanması için bir protokol açıklar. Ayrıca, gerçek-t&lt;40,000 hücre kullanılarak ime PCR deneyleri hücre düşük sayıda ilave düşük giriş RNA kitleri gerek kalmadan gerçekleştirilebilir bu gen analiz deneyleri gösteren, GD3 ve GM2 / GD2 sintaz genlerinin belirlenmesi için tarif edilmiştir.

Bu yazıda anlatılan protokol kültürlü kaliteli ve yapıştırılır dinlenme ve aktif insan naif CD4 + T hücreleri (Şekil 1) işler. Aktive edilmiş CD4 + T hücreleri durumda (Şekil 1A) kıyasla karakteristik bir proliferatif profil (Şekil 1B) göstermektedir. CD25 Geç aktivasyon işaretleme konfokal mikroskopi (Şekil 1C) tarafından gözlemlenen 72 saatte etkin aktivasyonunu değerlendi...

Watch this Scientific Journal Video about Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy at JoVE.com

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

Istirahat bölgesindeki gangliosidler ve aktive edilmiş insan naif CD4 + T hücrelerinin membran ekspresyonunu ve sınırlandırılmasını belirlemek için mikroskopla kullanılmak için bir standart antikor boyama protokolü tarif eder. Ayrıca gerçek zamanlı PCR deneyleri &lt;ek düşük giriş RNA kitleri gerekmez 40.000 hücreleri kullanarak tarif edilir.

CD4 hazırlanması<sup&gt; +</sup&gt; Mikroskopi GD3 ve GD2 Gangliozidleri Membran İfade Analizi T hücreleri

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

preparation-cd4-t-cells-for-analysis-gd3-gd2-ganglioside-membrane

Research

JoVE Journal

Immunology and Infection

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

8.4K Görüntüleme.  Universidad Autónoma del Estado de Morelos.  Istirahat bölgesindeki gangliosidler ve aktive edilmiş insan naif CD4 + T hücrelerinin membran ekspresyonunu ve sınırlandırılmasını belirlemek için mikroskopla kullanılmak için bir standart antikor boyama protokolü tarif eder. Ayrıca gerçek zamanlı PCR deneyleri 

Video: Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

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 ...

İmmünoloji ve Enfeksiyon

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

Diyabetojenik CD4 Kabul edilmiş Transfer ile Farelerde Hızlandırılmış Tip 1 Diyabet İndüksiyon + T Hücreleri

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Naif CD4 adenoviral İletimi Treg Farklılaşma Eğitim Hücreler T

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

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&#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 ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 Enfekte CD4 Doğuştan Algılama Değerlendirilmesi<sup&gt; +</sup&gt; Bir kullanılması plazmositoid dendritik hücreler tarafından T Hücreleri<em&gt; Ex vivo</em&gt; Co-kültür sistemi.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Efektör CD4 Evlatlık transferi ile Kemirgen Bağırsak Enflamasyon indüksiyonu<sup&gt; +</sup&gt; CD45RB<sup&gt; Yüksek</sup&gt; Bağışık yetmezliği olan farelere içine T Hücreleri

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

Iltihaplı Dermisinde içinde görüntüleme CD4 + T hücre Geçiş Göç

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Yapılandırılmış Aydınlatma Mikroskopi Verilerinin uzay-zamansal Görüntü Korelasyon Spektroskopisi nicel T Hücreleri kortikal Aktin Akışı

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

Bir TGF-β-içeren Protokolü kullanılarak naif CD4 + T hücrelerinden insan CD4 + FoxP3 + Bağlı regülatör T hücreleri (iTregs) in vitro ayrımında

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.