Name: generation of human cd40-activated b cells
Uploaded: 2009-10-16T23:30:00
Description: Watch this Scientific Journal Video about Generation of Human CD40-activated B cells Video at JoVE.com

<p class="jove_content"> PBMC insan CD40 aktive B hücreleri üretimi için protokol iki bölüme ayrılmıştır: Kısım A plaka bağlı besleyici hücreler olarak kullanılacaktır NIH/3T3 hücreleri, CD40 ligand ifade hazırlanmasında gösteriyor. Bölüm B, gerçek CD40-B kültür açıklar. </p><p class="jove_title"> A. besleyici hücreler Hazırlama (tCD40L NIH/3T3) </p><p class="jove_content"> TCD40L NIH/3T3 tamamen konfluent haline asla yapışkan bir fare fibroblast hücre hattı,. Hücreler bu nedenle haftada iki kez parçalı. Daha...

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	<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> <em style='font-style: italic'>J Clin Invest</em>. <b>100</b>, 2757-2765 (1997).</li><li>Schultze, J. L., Grabbe, S., vonBergwelt-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> <em style='font-style: italic'>Trends Immunol</em>. <b>25</b>, 659-664 (2004).</li><li>Bergwelt-Baildon, M. 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=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> <em style='font-style: italic'>Blood</em>. <b>107</b>, 2786-2789 (2006).</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> <em style='font-style: italic'>PLoS ONE</em>. <b>3</b>, 1464-14 (2008).</li><li>Urashima, M., Chauhan, D., Uchiyama, H., Freeman, G. J., Anderson, K. C. <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+triggered+interleukin-6+secretion+in+multiple+myeloma.">CD40 ligand triggered interleukin-6 secretion in multiple myeloma.</a> <em style='font-style: italic'>Blood</em>. <b>85</b>, 1903-1912 (1995).</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> <em style='font-style: italic'>Cancer Res</em>. <b>63</b>, 2836-2843 (2003).</li><li>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=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> <em style='font-style: italic'>Blood</em>. <b>99</b>, 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> <em style='font-style: italic'>Clin Exp Immunol</em>. <b>155</b>, 249-256 (2009).</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> <em style='font-style: italic'>Gene Ther</em>. <b>15</b>, 955-965 (2008).</li></ol>

<p class="jove_title"> A. Medya Hazırlanışı: </p><table border="1"cellspacing="0"><tbody><tr><td colspan="2"> <strong>Besleyici hücre vahşi tip orta</strong> </td><td colspan="2"> <strong>Besleyici hücre seçimi orta</strong> </td></tr><tr><td> <strong>Reaktif</strong> </td><td> <strong>Konsantrasyon</strong> </td><td> <strong>Reaktif</strong> </td><td> <strong>Konsantrasyon</strong> </td></tr><tr><td> DMEM-Ham / F12 </td><td></td><td> DMEM-Ham / F12 </td><td></td></tr><tr><td> L-Glutamine </td><td> 365 mg / ml </td><td> L-Glutamine </td><td> 365 mg / 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> G-418 </td><td> 0,7 mg / ml </td></tr></table><table cellspacing="0" border="1"><tbody><tr><td colspan="2"> <strong>CD40-B yıkama orta</strong> </td><td colspan="2"> <strong>CD40-B kültür ortamı</strong> </td></tr><tr><td> <strong>Reaktif</strong> </td><td> <strong>Konsantrasyon</strong> </td><td> <strong>Reaktif</strong> </td><td> <strong>Konsantrasyon</strong> </td></tr><tr></tr></tbody><tbody><tr><td> IMDM </td><td><br /></td><td> IMDM </td><td><br /></td></tr></tbody><tbody><tr><td> L-Glutamine </td><td> 584 mg / ml </td><td> L-Glutamine </td><td> 584 mg / ml </td></tr></tbody><tbody><tr><td> Hepes </td><td> 25 mM </td><td> Hepes </td><td> 25 mM </td></tr></tbody><tbody><tr><td> Gentamisin </td><td> 15 mcg / mL </td><td> Gentamisin </td><td> 15 mcg / mL </td></tr></tbody><tbody><tr><td><br /></td><td><br /></td><td> rh Transferrin </td><td> 50 mcg / mL </td></tr></tbody><tbody><tr><td><br /></td><td><br /></td><td> rh İnsülin </td><td> 5 mg / ml </td></tr></tbody><tbody><tr><td><br /></td><td><br /></td><td> AB-Humanserum </td><td> % 10 </td></tr></tbody></table><p class="jove_title"> B. Çeşitli Reaktifler: </p><table cellspacing="0" border="1"><tbody><tr><td> <strong>Reaktif</strong> </td><td> <strong>Şirket</strong> </td><td> <strong>Hiçbir sipariş</strong> </td></tr><tr></tr><tr><td> DMEM / Ham Kullanıcı F12 </td><td> PAA </td><td> Cat No: E15-813 </td></tr><tr><td> IMDM </td><td> Invitrogen Gibco ® </td><td> REF 21980-032 </td></tr><tr><td> Dulbecco&#39;nun PBS (10x) </td><td> PAA </td><td> Kedi No H15-011 </td></tr><tr><td> AB-İnsan Serum </td><td> Invitrogen </td><td> Kedi yok 34005100 </td></tr><tr><td> Holo-Transferrin insan </td><td> Sigma </td><td> Kedi yok T0665 </td></tr><tr><td> İnsülin insan </td><td> Sigma </td><td> Kedi yok I2643 </td></tr><tr><td> Gencin ® </td><td> Delta seçin </td><td> Art No 7395800 </td></tr><tr><td> Rekombinant İnsan İnterlökin-4 </td><td> Immunotools </td><td> Kedi yok 11130045 </td></tr><tr><td> Bir Siklosporin </td><td> Novartis </td><td> Kedi yok NDC 0078-0109-01 </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> G-418 Sülfat </td><td> PAA </td><td> Kedi No P02-012 </td></tr><tr><td> Tripsin / EDTA (10x) </td><td> Invitrogen Gibco ® </td><td> REF 15400-054 </td></tr></tbody></table><p class="jove_title"> C. Çeşitli Sarf Malzemeleri: </p><table cellspacing="0" border="1"><tbody><tr><td> <strong>Reaktif</strong> </td><td> <strong>Şirket</strong> </td><td> <strong>Hiçbir sipariş</strong> </td></tr><tr></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>

generation of human cd40-activated b cells

<p class="jove_content"> CD40-aktive B hücreleri (CD40-B hücreleri) immüno-uyarıcı antijen sunan hücreler (APC), kanser immünoterapi <sup>için</sup> 1-3 alternatif bir kaynak olarak tespit edilmiştir . CD40-B hücreleri birkaç farklı biyolojik ve teknik özellikleri Dendritik hücreler (DH), en iyi karakterize APC ile karşılaştırıldığında. DC&#39;ler benzer şekilde, B hücreleri MHC ve co-uyarıcı moleküller (Fig.1b) artmış ekspresyonu, interlökin-4 ve CD40 ligand (CD40L) ile stimülasyon sonra, güçlü bir göçmen kapasite ve mevcut antijen sunumu T hücrelerinin verimli sergilerler. Ancak, olgunlaşmamış ya da olgun DC&#39;ler aksine, CD40-B hücreleri CD62L, CCR7/CXCR4 oluşan tam bir lenf nodu homing üçlüsü ifade ve ikincil lenfoid organlara homing gerekli lökosit fonksiyon antijen-1 (LFA1, CD11a/CD18) (Fig.1a) <sup>3</sup> CD40-B hücreleri, periferik kanda çok az miktarda daha yüksek saf CD40 B hücreleri sağlıklı donörlerden (&gt; <sup>10</sup> 9 hasta başına hücreleri) yanı sıra kanser çok büyük <em>miktarda in</em> vitro açılabilir zorluklar olmadan oluşturulabilir hasta (Fig.1c d) <sup>1,4.</sup> </p><p class="jove_content"> Bu protokol, insan hücre tamamen aktif CD40-B hücreleri nasıl elde göstermektedir. Hücre kültürü için anahtar moleküller CD40 ligand, interlökin-4 (IL-4) ve siklosporin A (CsA), 3-4 günde bir kültür döngüsü yenileniyor. Laboratuvar amaçları için CD40 stimülasyon, rekombinant insan CD40 ligand (tCD40L <sup>NIH/3T3)</sup> 5 ifade NIH/3T3 hücreleri tarafından sağlanmaktadır . -Transfekte olmayan hücreleri ile kirlenmesini önlemek için, transfektanları insan CD40 ligand ekspresyonu (Şekil 2) düzenli olarak kontrol edilmelidir. </p><p class="jove_content"> 14 gün sonra CD40 B hücre kültürleri fazla% 95 saf B hücreleri CD40-B hücreleri ve 65 gün içinde bir genişleme sık fonksiyonu <sup>1, 4</sup> herhangi bir kayıp olmaksızın mümkün oluşur. CD40-B hücreleri verimli, süreç kadar sürebilir ve T hücrelerinin mevcut antijenler <sup>6.</sup> Sadece başbakan naϊve değil, aynı zamanda bellek T hücreleri <sup>7,8</sup> genişletmek. CD40 ile aktive olan B hücreleri, B-hücre aktivasyonu, farklılaşma ve fonksiyon çalışma için kullanılabilir. Ayrıca, tümörler <sup>9</sup> karşı tedavi veya önleyici aşı için umut verici bir aracı temsil eder. </p>

Watch this Scientific Journal Video about Generation of Human CD40-activated B cells Video at JoVE.com

Generation of Human CD40-activated B cells Video (Video) | JoVE

<p class="jove_content">Bu video mevcut<em&gt; Ex vivo</em&gt; Insan CD40 aktive B hücreleri, CD40 ligand ve interlökin-4 ile stimülasyon periferik kan mononükleer hücreleri (hücre) üretimi ve genişleme (CD40-B).</p>

İnsan CD40 aktive B hücreleri üretilmesi

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

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Generation of Human CD40-activated B cells

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