Biz lütfen Dr. Clemens Wendtner tmuCD40 Ligandla HeLa hücre hattı sağlamak için teşekkür ederiz.

Splenositlerin fare CD40 aktive B hücreleri (mCD40B) üretimi için protokol iki bölüme ayrılmıştır: Kısım A olarak kullanılacak plaka HeLa hücreleri (tmuCD40L HeLa), ifade fare CD40 ligand (CD40L) hazırlanmasında gösteriyor. besleyici hücreler bağlı. Bölüm B, gerçek bir fare CD40-B kültür açıklar. A. besleyici hücreler Hazırlama (tmuCD40L HeLa) TmuCD40L HeLa tamamen konfluent haline asla yapışık bir insan epitel hücre hattı,. Hücreler bu nedenle haftada iki kez parçalı. Daha fazla 6 hafta içinde Kültürleme tavsiye edilmez. <ol><li> Steril bir pipet ile primer kültür eski orta çıkarın ve 10 ml 1x PBS ile yıkayın hücreleri. PBS ile yıkandıktan sonra aspire. </li><li> 37, 10-15 dakika için 75 cm 2 balon 4 ml 1x tripsin / EDTA ekleyin ° C. Hücreleri ayırmak için hafif dokunarak kullanın. </li><li> Vahşi tip orta 10 ml ekleyin ve tripsin ile sindirim durdurmak için yavaşça dönebilen. </li><li> Hücre süspansiyonu steril bir pipet yardımıyla 50 ml tüp içine aktarın ve 7 dakika boyunca 265 xg&#39;de hücreleri aşağı döndürün. </li><li> Süpernatantı ve vahşi tip orta 10 ml hücre pelletini tekrar süspansiyon haline getirin. Hücre süspansiyonu bir kısım hücre sayısı sayın ve uygun sayıda hücre ile üç 50 ml tüpler hazırlamak: <ol><li> Altkültürün 2.5 x 10 6 hücre </li><li> Fare CD40 B hücre kültürü için kullanılan / iyi ışınlanması için 0.4 x 10 6 hücre </li><li> dondurmak için geri kalan (gerekirse). </li></ol></li><li> 7 dakika boyunca 265 xg&#39;de hücreleri aşağı spin. </li><li> Süpernatantı. <ol><li> Alt kültüre için tekrar süspansiyon 2.5 ° C ile% 5 CO 2 75 cm 2 hücre kültürü şişesi 10 ml seçim ortamda (hücre yoğunluğu 2.5 x 10 5 hücre / ml) ve 37 hücreler inkübasyon x 10 6 hücre. Haftada iki kez hücreleri bölün. </li><li> Fare CD40 B hücre kültürü için bir adet 6-plaka için 2,4 x 10 6 hücre gerekir. 0.2 x 10 6 hücre / mL yoğunluğu vahşi tip orta tmuCD40L HeLa hücreleri yeniden süspanse edin ve 78 Gy ışın tedavisi. Yanı sıra, her 37 ° inkübe ° C ile% 5 CO 2 hücre süspansiyonu Plaka 2 mL. TmuCD40L HeLa hücrelerinde yapışık B-hücre uyarılması için hazırlanmış tabak kullanın (en az 4 saat: mikroskop kullanarak bağlılık kontrol edin; B-hücre stimülasyon başlatmak için 24 saat daha beklemek gerekmez). (B. devam) </li></ol></li></ol> B. murin CD40 B hücre kültürü CD40 stimülasyon (gün 0) splenositlerin I. Hazırlık: Lütfen dikkat: besleyici hücreler yapışık olduğu tespit devam etmeden önce. Her zaman fare interlökin-4, β-mercaptoethanol ve büyüme ortamına siklosporin, kullanımdan hemen önce taze çözümler ekleyin. <ol><li> C57BL / 6 farelerinde (H-2b haplotip), dalak inceleyin ve 10-20 ml DMEM iki kez yıkayın, başka bir tüp 10ml pipet ile transfer Gentamisin [15μg/mL] ile desteklenmiş. Fare transfer kirleticiler (örneğin saç) kaçının. (100μm) bir hücre süzgecinden geçirilerek dalak Kıyma ve orta başka bir 10 mL süzgeç durulayın. Oda sıcaklığında 7 dakika süreyle 265 xg&#39;de splenositlerin Spin, 7ml muCD40-B hücre, orta ve Ficoll yoğunluğu santrifüj ayrı lenfositlerin tekrar süspansiyon haline getirin. 5 ml, 30 dakika için 450 xg&#39;de 15 mL tüp ve santrifüj Fare-Pancoll (oda sıcaklığında önceden ısıtılmış) tabakası hücreleri oda sıcaklığında frensiz yapmak için. </li><li> Arayüzü rahatsız lenfosit tabakası bırakarak, en üst katmanı çizin. Taze bir 50 ml tüp lenfosit katmanı Transferi, diğer hücreleri çıkarmak için 7 dakika süreyle 265 xg&#39;de aşağı iplik Gentamisin [15μg/mL] ile 30ml DMEM ile bir kez yıkayın. Süpernatantı atın ve mCD40-B yıkama orta 10 ml hücreleri tekrar süspansiyon haline getirin. Hücre süspansiyonu bir kısım hücre sayısını belirler. Hücre süspansiyonu bir kısım sayarak hücre sayısını belirler. </li><li> 7 dakika boyunca 265 xg&#39;de gerekli miktarda hücre aşağı spin. 6-iyi bir plaka için 5 x 10 6 hücre / kuyu, böylece plaka başına 30 x 10 6 hücre ihtiyaç vardır. </li><li> Süpernatantı ve büyüme faktörü olarak, siklosporin 100 mcM β-mercaptoethanol ve 0.63 mcg / ml önlemek için 1.25 x 10 6 hücre / ml taze interlökin-4 1 U / ml ile desteklenmiş mCD40-B kültür ortamı lenfositlerin tekrar süspansiyon T-hücrelerinin akıbet (Verilen konsantrasyonları bir ml kültür ortamı bakın!). </li><li> 6 plaka tmuCD40L HeLa hücreleri ile ön inkübasyona süpernatantı. </li><li> 6 plaka her bir kuyu için 4 ml (1.25 x 10 6 hücre / ml) lenfosit süspansiyonu yavaşça ekleyin. </li><li> 37 ° ° C,% 5 CO 2 plaka inkübe . </li&gt; <li> 3. günde hücreleri (II.1 ile devam edin.) Reculture. </li></ol> II. MCD40B hücreleri (3. gün ve daha sonra haftada iki kez) altkültürünün: <ol><li> 50 ml tüp içine 10 ml pipet ve havuz onları şiddetle yukarı ve aşağı pipetleme Hasat mCD40B hücre kümesi. </li><li> 7 dakika boyunca 265 xg&#39;de Spin ve süpernatant tamamen mCD40-B yıkama orta yerine. Bir kısım hücre miktarını sayma, 7 dakika boyunca 265 xg&#39;de hücreleri aşağı doğru döndürün. 0.75 x 10 6 hücre / ml &#39;lik bir konsantrasyon mCD40-B kültür ortamında süspanse edin mCD40B hücreleri. </li><li> 1 U / ml, 100 mcM β-mercaptoethanol ve orta siklosporin 0.63 mcg / ml &#39;lik bir konsantrasyon interlökin-4 fare taze çözümler ekleyin. </li><li> 6 plaka ışınlanmış tmuCD40L HeLa hücreleri önceden inkübe süpernatantı. </li><li> 6 plaka her iyi mCD40B hücre süspansiyonu (0.75 x 10 6 hücre / ml) 4 mL yavaşça ekleyin. </li><li> 37 ° plakaları inkübe ° C,% 5 CO 2 . </li><li> Yine her 3-4 günde bir toplam 14 gün sonra yüksek saflıkta fare CD40 aktive B hücreleri ile sonuna kadar hücreleri altkültürü. </li></ol> C. Sorun Giderme: CD40-B hücreleri büyümek ne olur değil mi? <ol><li> Besleyici hücreler CD40 ligand ifade için kontrol ettiniz? </li><li> 24 saat daha eski uyarılması için kullanılan plaka bağlı besleyici hücreler musunuz? </li><li> Mycoplasma ile bir kirlenme var mı? </li><li> Takviyesi için kullanılan interlökin-4 çözüm taze çözülmüş ve uygun biyolojik aktivite var mı? </li><li> Β-mercaptoethanol ve siklosporin A doğru konsantrasyon eklendi? </li></ol><img src="/files/ftp_upload/1734/1734fig1a.jpg" alt="Şekil 1" /> Şekil 1a. <img src="/files/ftp_upload/1734/1734fig1b.jpg" alt="Şekil 1 b" /> Şekil 1b. <img src="/files/ftp_upload/1734/1734fig1d.jpg" alt="Şekil 1 d" /> Şekil 1d. <img src="/files/ftp_upload/1734/1734fig2.jpg" alt="Şekil 2" /> Şekil 2.

<ol>
	<li>Schultze, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+human+B+cells:+an+alternative+source+of+highly+efficient+antigen+presenting+cells+to+generate+autologous+antigen-specific+T+cells+for+adoptive+immunotherapy.">CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy.</a> J Clin Invest. 100 (11), 2757-2765 (1997).</li><li>Wiesner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+immortalization+of+human+B+cells+by+CD40+ligation.">Conditional immortalization of human B cells by CD40 ligation.</a> PLoS ONE. 3, e1464-e1464 (2008).</li><li>Kondo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+antigen-specific+cytotoxic+T+cells+using+retrovirally+transduced+CD40-activated+B+cells.">Efficient generation of antigen-specific cytotoxic T cells using retrovirally transduced CD40-activated B cells.</a> J Immunol. 169 (4), 2164-2171 (2002).</li><li>Liebig, T. M., Fiedler, A., Zoghi, S., Shimabukuro-Vornhagen, A., Bergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+CD40-activated+B+cells.">Generation of human CD40-activated B cells.</a> J Vis Exp. 32, (2009).</li><li>von Bergwelt-Baildon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+express+full+lymph+node+homing+triad+and+induce+T-cell+chemotaxis:+potential+as+cellular+adjuvants.">CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants.</a> Blood. 107 (7), 2786-2789 (2006).</li><li>Lapointe, R., Bellemare-Pelletier, A., Housseau, F., Thibodeau, J., Hwu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-stimulated+B+lymphocytes+pulsed+with+tumor+antigens+are+effective+antigen-presenting+cells+that+can+generate+specific+T+cells.">CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells.</a> Cancer Res. 63 (11), 2836-2843 (2003).</li><li>Ahmadi, T., Flies, A., Efebera, Y., Sherr, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+Ligand-activated,+antigen-specific+B+cells+are+comparable+to+mature+dendritic+cells+in+presenting+protein+antigens+and+major+histocompatibility+complex+class+I-+and+class+II-binding+peptides.">CD40 Ligand-activated, antigen-specific B cells are comparable to mature dendritic cells in presenting protein antigens and major histocompatibility complex class I- and class II-binding peptides.</a> Immunology. 124 (1), 129-140 (2008).</li><li>Bergwelt-Baildon, M. S. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+primary+and+memory+cytotoxic+T+lymphocyte+responses+are+efficiently+induced+by+means+of+CD40-activated+B+cells+as+antigen-presenting+cells:+potential+for+clinical+application.">Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application.</a> Blood. 99 (9), 3319-3325 (2002).</li><li>Kondo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+can+be+generated+in+high+number+and+purity+in+cancer+patients:+analysis+of+immunogenicity+and+homing+potential.">CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential.</a> Clin Exp Immunol. 155 (2), 249-256 (2009).</li><li>Schultze, J. L., Grabbe, S., von Bergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DCs+and+CD40-activated+B+cells:+current+and+future+avenues+to+cellular+cancer+immunotherapy.">DCs and CD40-activated B cells: current and future avenues to cellular cancer immunotherapy.</a> Trends Immunol. 25 (12), 659-664 (2004).</li><li>Mayr, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+translocations+are+associated+with+poor+prognosis+in+chronic+lymphocytic+leukemia.">Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia.</a> Blood. 107 (2), 742-751 (2006).</li><li>Mason, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-loaded+CD40-activated+B+cells+stimulate+antigen-specific+T-cell+responses+in+dogs+with+spontaneous+lymphoma.">RNA-loaded CD40-activated B cells stimulate antigen-specific T-cell responses in dogs with spontaneous lymphoma.</a> Gene Ther. 15 (13), 955-965 (2008).</li></ol>

Deneylerde laboratuar hayvanları tutmak için ulusal ve Avrupa kurallarına uygun olarak yapıldı. Alman hayvan refahı hukuk değinen hayvanların uygun yetkililer tarafından düzenli olarak kontrol altına alındı.

Bu protokolün, özellikle de verimli nesil fare CD40-aktive B hücreleri verimli CD40-uyarıcı ve kullanılan fare gerginlik türü, kökeni ile ilgili Değişiklikler mümkündür. Ahmedi et al. CD40 ligand fare ile transfekte fare fibroblastlar için benzer sonuçlar vermiştir. Xeno-antijen Bu bağlamda sunumda kesin olarak hariç, ama güvenlik ve in vivo toksisite ile ilgili yoğun çalışmalar insan transfekte HeLa hücreleri kullanarak bu ayarı inflamasyon veya oto-bağışıklık reaksiyonu için hiçbir belirti verdi olabilir . Ancak, en iyi alternatif, bildiğimiz kadarıyla piyasada mevcut değil, aynı zamanda, faaliyet aktive ve prolifere CD40 ligand çözünen bir fare temsil eder. Bu protokol aynı zamanda 129S2/SvPas fareler (haplotype H-2b) lenfositleri için çalışır, ancak şimdiye kadar, daha fazla fare suşları için henüz değerlendirilmemiştir. Ayrıca hücre yoğunluğu ayarı farklılıkları, sitokin konsantrasyonu, süresi siklosporin A tedavisi, hücre kültürü medya ve yemekleri de benzer çoğalması oranları ile fare B hücreleri verimli CD40 etkinleştirme yol açabilir. Ancak, yoğun çalışma, mevcut sistem aktivasyonu ve çoğalması oranlarını maksimize bu sistem optimize harcanmıştır.

A. Medya Hazırlanışı: <table border="1"><tbody><tr><td> Besleyici hücre vahşi tip orta </td><td> Besleyici hücre seçimi orta </td><td></td></tr><tr><td> Reaktif </td><td> Konsantrasyon </td><td> Reaktif </td><td> Konsantrasyon </td></tr><tr><td> RPMI 1640 </td><td></td><td> RPMI 1640 </td><td></td></tr><tr><td> L-Glutamine </td><td> 300 mcg / mL </td><td> L-Glutamine </td><td> 300 mcg / mL </td></tr><tr><td> FBS </td><td> % 10 </td><td> FBS </td><td> % 10 </td></tr><tr><td> Hepes </td><td> 10 mM </td><td> Hepes </td><td> 10 mM </td></tr><tr><td> Gentamisin </td><td> 15 mcg / mL </td><td> Gentamisin </td><td> 15 mcg / mL </td></tr><tr><td></td><td></td><td> Hygromycin B </td><td> 0.2 mg / ml </td></tr></table><table border="1"><tbody><tr><td> mCD40-B yıkama orta </td><td> mCD40-B kültür ortamı </td></tr><tr><td> Reaktif </td><td> Konsantrasyon </td><td> Reaktif </td><td> Konsantrasyon </td></tr><tr></tr><tr><td> D-MEM </td><td> </td><td> D-MEM </td><td> </td></tr><tr><td> L-Glutamine </td><td> 580 mg / ml </td><td> L-Glutamine </td><td> 580 mg / ml </td></tr><tr><td> Glikoz </td><td> 4,5 mg / ml </td><td> Glikoz </td><td> 4,5 mg / ml </td></tr><tr><td> Hepes </td><td> 10 mM </td><td> Hepes </td><td> 10 mM </td></tr><tr><td> Gentamisin </td><td> 15 mcg / mL </td><td> Gentamisin </td><td> 15 mcg / mL </td></tr><tr><td> </td><td> </td><td> MEM </td><td> 0.1mm (1x) </td></tr><tr><td> </td><td> </td><td> FBS </td><td> % 10 </td></tr></tbody></table> B. Çeşitli Reaktifler: <table border="1"><tr><td> Reaktif </td><td> Şirket </td><td> Hiçbir sipariş </td></tr><tr><td> RPMI 1640 </td><td> Invitrogen Gibco </td><td> REF 21.875 </td></tr><tr><td> D-MEM </td><td> Invitrogen Gibco </td><td> REF 41.965 </td></tr><tr><td> Dulbeccos PBS (10x) </td><td> PAA </td><td> Kedi No H15-011 </td></tr><tr><td> Gencin </td><td> Delta seçin </td><td> Art No 7395800 </td></tr><tr><td> FBS </td><td> Lonza </td><td> Kedi yok DE14-802C </td></tr><tr><td> Hepes Tampon </td><td> PAA </td><td> Kedi yok S11-001 </td></tr><tr><td> MEM NEAA </td><td> Invitrogen Gibco </td><td> REF 11140 </td></tr><tr><td> Hygromycin B </td><td> PAA </td><td> Kedi No P02-015 </td></tr><tr><td> Rekombinant murin İnterlökin-4 </td><td> Immunotools </td><td> Kedi yok 12340045 </td></tr><tr><td> Bir Siklosporin </td><td> Novartis </td><td> Kedi yok NDC 0078-0109-01 </td></tr><tr><td> Tripsin / EDTA (10x) </td><td> Invitrogen Gibco </td><td> REF 15400-054 </td></tr></table> C. Çeşitli Sarf Malzemeleri: <table border="1"><tbody><tr><td> Reaktif </td><td> Şirket </td><td> Hiçbir sipariş </td></tr><tr><td> Steril pipet uçları </td><td> Sarstedt </td><td></td></tr><tr><td> 6-plaka </td><td> Nunc </td><td> Kedi yok 140.675 </td></tr><tr><td> 50ml konik tüp </td><td> BD Falcon ™ </td><td> Kedi yok 352.070 </td></tr><tr><td> Doku kültürü şişesi </td><td> Sarstedt </td><td> Kedi yok 83.1813.002 </td></tr></tbody></table>

murine model of cd40-activation of b cells

B hücreleri üzerinde araştırma CD40 aktivasyonu ve antijen sunumu kapasitesi iyileştirdiğini göstermiştir. Interlökin-4 ve CD40 ligand (CD40L) ile uyarıldığında, insan B hücreleri, 14 gün içinde son derece saf CD40-B hücreleri (çok büyük miktarda küçük miktarlarda periferik kan zorluklar olmadan açılabilir&gt; 10<sup&gt; 9</sup&gt; Hasta başına kanser hastalarının yanı sıra sağlıklı donörlerin hücrelerinin)<sup&gt; 1-4</sup&gt;. CD40-B hücreleri önemli lenf nodu homing molekülleri ifade ve T hücreleri çekebilecek<em&gt; In vitro</em<sup&gt; 5</sup&gt;. Ayrıca verimli T hücrelerine, süreç ve mevcut antijenlere kadar sürebilir<sup&gt; 6,7</sup&gt;. CD40-B hücreleri sadece başbakan naif değil gösterilen, aynı zamanda bellek T hücreleri genişletmek<sup&gt; 8,9</sup&gt;. Bu nedenle, CD40-aktive B hücreleri (CD40-B hücreleri) bir alternatif kaynak olarak çalışılmıştır immüno-uyarıcı hücre tabanlı immunotherapy1, 5,10 antijen sunan hücreler (APC). CD40-B hücreleri in vivo etkili T hücre yanıtları uyardığı ve fare CD40 aktive B hücreleri üretimi için bir hücre kültürü sistemi kurdu yatan mekanizma çalışma konusunda daha fazla çalışma için. Splenositlerin kullanılması veya CD40 aktivasyonu için C57BL / 6 fareler B hücreleri saflaştırılmış, optimal koşullar aşağıdaki gibi tespit edilmiştir: C57BL / 6 farelerinde (H-2b haplotip) lenfositler dalak itibaren yoğunluk gradiyenti santrifüj ve HeLa ile birlikte kültürlü arıtılmış rekombinant fare CD40 ligand (tmuCD40L HeLa) ifade hücreleri<sup&gt; 11</sup&gt;. Hücreler, 3-4 günde bir recultured ve CD40L, interlökin-4 gibi temel bileşenleri, mercaptoethanol ve siklosporin A takviye edilir. Bu protokolde insan CD40-B hücreleri (Şekil 1a, b) APC-fenotip benzer tam aktif fare CD40-B hücreleri (mCD40B) nasıl elde göstermektedir. CD40 stimülasyon hücre kültürü 14 gün içinde hızlı bir sonucudur ve yüksek saflıkta (&gt;% 90) CD19 + B hücreleri genişleme (Şekil 1c, d) yol açar. -Transfekte olmayan hücreleri ile kirlenmesini önlemek için, transfektanları fare CD40 ligand ekspresyonu (Şekil 2) düzenli olarak kontrol edilmesi. Murin CD40 aktive B hücreleri in vitro ve in vivo olarak APC olarak çalışması için kendi potansiyelini araştırmak için B-hücre aktivasyonu ve farklılaşması yanı sıra çalışma kullanılabilir. Ayrıca, tümörlere karşı tedavi veya önleyici aşı oluşturulması için umut verici bir aracı temsil eder ve bu yaklaşımın immünojenite ve güvenlik ile ilgili sorulara cevap vermeye yardımcı olacak<sup&gt; 12</sup&gt;.

Watch this Scientific Journal Video about Murine Model of CD40-activation of B cells at JoVE.com

Murine Model of CD40-activation of B cells

Bu video, CD40 aktivasyonu ve dokunulmazlık indüksiyonu eğitim için bir model antijen sunan hücre (APC) olarak da kullanılabilir C57BL / 6 farelerin dalak, fare B hücreleri genişleme prosedürü göstermektedir.

Murin Model B hücreleri CD40 aktivasyonu

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

murine-model-of-cd40-activation-of-b-cells

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.6K Görüntüleme.  University Hospital of Cologne. Bu video, CD40 aktivasyonu ve dokunulmazlık indüksiyonu eğitim için bir model antijen sunan hücre (APC) olarak da kullanılabilir C57BL / 6 farelerin dalak, fare B hücreleri genişleme prosedürü göstermektedir.

Video: Murine Model of CD40-activation of B cells

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

Sınıf Anahtarı Rekombinasyon murin B Hücreleri Saf İndüksiyon ve Değerlendirme

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

İmmünoloji ve Enfeksiyon

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

B Hücreleri Rekombinant Retroviral Üretim ve Enfeksiyon

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Alerjen Bağlı Astım murin Modeli

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

Mürin Norovirüs için Plak Testi

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Doğru Algılama Fare B Lineage Hücreleri IgE-ifade

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Ölümsüzleştirilmiştir Sıralama B hücrelerinden Rekombinant İnsan IgG Monoklonal Antikorlarının Üretimi

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B Grubu bir murin modelinde<em&gt; Streptococcus</em&gt; Vajinal Kolonizasyon

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

Fenotip ve Fonksiyon Analizi için algılama ve Nadir zenginleştirilmesi antijene özgü B hücreleri

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Yamultma akış kaynaklı geçiş antikorundan marjinal bölge B hücrelerinin in Vitro analizine

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or mucosa and ensure humoral immunity. Due to their low frequency and location, the study of PCs is difficult in human. We reported a B to PC in vitro differentiation model using selected combinations of cytokines and activation molecules that allow to reproduce the sequential cell differentiation occurring in vivo. In this in vitro model, memory B cells (MBCs) will differentiate into pre-plasmablasts (prePBs), plasmablasts (PBs), early PCs and finally, into long-lived PCs, with a phenotype close to their counterparts in healthy individuals.&#160;We also built an open access bioinformatics tools to analyze the most prominent information from GEP data related to PC differentiation. These resources can be used to study human B to PC differentiation and in the current study, we investigated the gene expression regulation of epigenetic factors during human B to PC differentiation.

Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation model.

Uzun ömürlü plazma hücreleri insan Normal bellek B hücrelerine vitro farklılaştırma modelinde

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...