DRW NIH hibe AI089972 ve AI113217 tarafından ve Mukozal İmmünoloji Çalışmaları Ekibi tarafından desteklenen ve Burroughs Wellcome Fonu Tıbbi Bilimciler için Kariyer Ödülü tutar.

NOT: Burada açıklanan tüm deneyler Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) kurallarına uygun olarak ve Hayvancılık Araştırma Hastanesi Çocuk (ARCH), Boston, Mass tarafından onayladı. Reaktifler 1. Hazırlık <ol><li> 1,000x, IL-4 Stok: H 2, O ya da% 0.1 bovin serum albümini (BSA) içinde 20 ug / ml olacak şekilde seyreltin, IL-4, sitokin. -20 ° C&#39;de 1.5 ml mikrosantrifüj tüplerine ve saklamak için 200 ul alikolar içinde dağıtın. </li><li> 1,000x anti-CD40: 4 ° C &#39;de, 1.0 mg / ml arasında, mağazada üreticisinden elde edilir. </li><li> B hücre uyarılmasını ortamı: RPMI-1640 ortamı içinde 425 ml&#39;lik, ekleme 75 taze çözülmüş fetal buzağı serumu mi (FCS), 1.0 M HEPES tamponunda, 5 ml L-glutamin stokunun 10 mi, 5 ml Penisilin: Streptomisin stoku, 5 2-merkaptoetanol mi Asgari Temel Ortam Aslî Olmayan Amino Asitler (MEM-NEAA) ve 3.5 ul. Bir haftaya kadar 4 ˚C Ortamı tutun. IL-4 (20 ng / ml) ve anti-CD40 (1.0 ug / ml), kullanılmadan hemen önce eklemesadece gerekli ortamın hacmi. </li><li> % 2 FCS-FACS Tampon: 1x PBS 490 ml FCS, 10 ml ilave edilir. 4 ° C&#39;de tutun. </li><li> 2x tripsin-EDTA çözeltisi: 1x PBS, 20 ml 10 x tripsin-EDTA stok çözeltisi (5.0 gr, tripsin, litre başına 2.0 g EDTA) 5 ml seyreltin. Mağaza tripsin-EDTA 2x 4 ° C&#39;de alikotları çalışma. Kullanmadan önce oda sıcaklığına ısınmasına izin verin. </li><li> Cenin buzağı serumu: en fazla iki hafta boyunca 4 ° C&#39;de eritilmiş FCS tutun. Sitoplazmik boyama prosedürü önce buz 15 dk yerleştirin. </li><li> Nötr-tamponlu formalin:% 10 formalin solüsyonu (% 3.7 paraformaldehid) hazırlayın. Formalin toksik ve uygun KKD giyerek bu nedenle laboratuar davlumbaz altında tutun. Bir kimyasal depolama kabini oda sıcaklığında saklayın formalin. </li><li> Metanol: -20 ° C&#39;de saklayın mutlak metanol (% 100) hücreleri geçirgenleştirildi hazır olana kadar. Bu, metanol yanıcı ve uygun bir dondurucuda saklanabilir gerektiğine dikkat ediniz. </li></ol> 2.Saflaştırılması ve B hücrelerinin aktivasyonu <ol><li> Taze hasat fare dalak kaynaktan, dikkatli bir şekilde 3 ml lik bir enjektör pistonuna sahip 70 mikron hücre süzgecinden organı basarak tek bir hücre süspansiyonu hazırlanır. 15 ml konik santrifüj tüpü içinde, 4 ° C&#39;de 5 dakika boyunca 300 x g&#39;de 1x 10 ml PBS hücrelerin ve santrifüj toplar. </li><li> 5 dakika boyunca alyuvar parçalanması Tamponu, 1 ml yüzer madde ve tekrar süspansiyon pelet süzün. 5 dakika boyunca 300 xg&#39;de 13.5 1x PBS ml ve santrifüj ile nötralize edin. </li><li> (İsteğe bağlı) Manyetik üreticinin talimatlarına göre MACS arıtma protokolü takip edilerek, boncuklar, anti-CD45R (B220) ile B hücreleri -etiketli ayırın. </li><li> 37 ° C &#39;de, IL-4 (20 ng / ml) ve 5 gün boyunca bir anti-CD40 (1.0 ug / ml) ihtiva eden ısıtılmış B hücre uyarılmasını ortam içinde 1.0 x 10 6 hücre / ml&#39;de, saflaştırılmış B hücrelerini stimüle eder. 6 oyuklu bir kültür plakasına (4 mL) içinde 2 kuyu ayrılmıştır kültür 8 ml ile başlar. </li><li> Cultu edin1.0 x 10 6 hücre / ml&#39;de korumak için günlük olarak tekrar. Diğer kuyulara bölme ve taze IL-4 ve anti-CD40 ihtiva eden B hücre uyarılmasını ortamı ile kültür seyreltilerek konsantrasyonu ayarlayın. </li></ol> 3. tripsinizasyonu, Fiksasyon ve Permeabilization <ol><li> Trypsinization <ol><li> 5 dakika boyunca 300 xg&#39;de, 15 ml konik tüp içinde santrifüj kültür hücrelerinin kadar 1.0 x 10 7 toplayın, ve süpernatant süzün. </li><li> Hücre toplama ortamından artık proteinin ayrılması için 10 ml PBS ile yıkayın. Santrifüj adımı tekrarlayın ve süpernatant süzün. </li><li> (Yaklaşık 100 ul) boşaltılarak sonra rezidüel hacim hücre pelletini. </li><li> Dikkatlice hücreler, oda sıcaklığında,% 0.1 (2x) tripsin-EDTA, 800 ul ilave edin. Konik tüp tarafında aşağı 45 ° açıyla yavaşça koyun. </li><li> Tüp kapatın ve sıvı tüp 5-6 kez uzunluğu boyunca uzanan bu yüzden tüp eğerek hafifçe karıştırıns. Bir tel, beyaz bir çökelti çözeltisi içinde meydana gelebilir. Bir dakika altındaki trypsinization zamanı sınırlayın. </li><li> Soğuk FCS, 1 ml tripsinizasyon reaksiyonu söndürün. Hemen soğuk PBS 10 ml ilave etmek suretiyle FCS ile seyreltilir. </li><li> 4 ° C&#39;de 5 dakika boyunca 300 x g&#39;de santrifüj, ardından süpernatan süzün ve buz tüpleri döndürür. </li></ol></li><li> Tespit <ol><li> Boşaltılarak sonra rezidüel hacim hücrelerin tekrar. Gerekirse, soğuk PBS ile 100 ul hacmi getirmek. </li><li> Bir çeker ocak içinde, hücrelere% 10 nötr tamponlu formalin çözeltisi 800 ul ve yukarı ve aşağı pipetleme ve. Tüp formalin ilavesi üzerine sıcak olacak. 10 dakika boyunca 37 ° C&#39;de inkübe edilir. </li></ol></li><li> Permeabilizasyon <ol><li> Hafif sallayarak hızında bir tezgah üstü vorteks karıştırıcı ayarlayın. </li><li> 10 ml&#39;lik bir serolojik pipet içine soğuk (-20 ° C) metanol içinde 9 ml çizin. </li><li> Vorteks mikser üzerinde hücreleri içeren tüp tutun ve yavaşça sallıyor başlangıçg kadar hücre. </li><li> Tüp hala çok titriyor metanol sürekli karıştırma olduğunu iken hücreleri üzerine damla damla düşer, böylece tüp soğuk metanol ekleyin. Metanol ilk 2 ml sonra dağıtma hızı yavaş bir akışla kadar arttırılabilir. </li><li> 30 dk permeabilization tamamlamak için tüp buz üzerinde bekletin. Seçenek olarak ise, hücreler, bir aya kadar -20 ° C&#39;de muhafaza edilebilir. </li></ol></li></ol> IgG1 ve IgE sınıf değiştirme 4. Floresan Boyama <ol><li> Santrifüj 5 dakika boyunca 300 xg&#39;de örnek ve metanol çözüm süzün. Beyaz bir çökelti hücreleri ile pelet olabilir. </li><li> 5 dakika Hücreleri yıkamak için 300 x g&#39;de oda sıcaklığında PBS 1x ve santrifüj 10 ml ilave edilir. Kez tekrarlayın. Not: oda sıcaklığı PBS ile pelet yıkanması adım 4.1 sonra oluşabilecek ekstra çökelti çözer. </li><li> % 2 FCS-FACS tamponu 10 ml ek yıkama yapın. </li><li> Yuvarlak tabanlı plaka 96 çukurlu numuneleri dağıtmave 5 dakika boyunca 300 x g&#39;de santrifüj. Süpernatant süzün ve buz üzerinde tabak koydu. </li><li> Aşağıdaki antikor seyreltmeleri ile oyuk başına% 2 FCS-FACS tamponu 60 ul Leke: Anti-IgM RPE / Cy7, 1: 200, anti-lgG1 PE, 1: 600, anti-IgE FITC, 1: 100, Anti- CD45R (B220) PerCP Cy5.5 @, 1: 200 </li><li> Boyama 5 dakika sonra, 300 x g, 5 dakika boyunca% 2 FCS-FACS tamponu ve santrifüj 150 ul plakayı yıkayın. </li><li> 5 ml&#39;lik bir FACS tüpü içine, bir 40 um naylon hücre süzgecinden% 2 FCS-FACS tamponu ve pipet 400 ul içinde süspanse. </li></ol> 5. Aşağıdaki Yolluk Strateji kullanın: <ol><li> FSC / SSC arsa (Şekil 1) bulunan patlatma lenfosit nüfus CD45R (B220) pozitif hücreleri izole. </li><li> B220 pozitif hücrelerden, ilgili popülasyonları ortaya IgE ve IgG1 arsa kullanın. </li></ol>

<ol>
	<li>Chaudhuri, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+immunoglobulin+heavy+chain+class+switch+recombination+mechanism.">Evolution of the immunoglobulin heavy chain class switch recombination mechanism.</a> Advances in immunology. 94, 157-214 (2007).</li><li>Gould, H. J., Sutton, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgE+in+allergy+and+asthma+today.">IgE in allergy and asthma today.</a> Nature reviews. Immunology. 8, 205-217 (2008).</li><li>Winter, W. E., Hardt, N. S., Fuhrman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunoglobulin E. Archives of Pathology &amp; Laboratory Medicine. 124 (9), 1382-1385 (2000).</li><li>Ozcan, E., Notarangelo, L. D., Geha, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+immune+deficiencies+with+aberrant+IgE+production.">Primary immune deficiencies with aberrant IgE production.</a> Journal of Allergy and Clinical Immunology. 122, 1054-1062 (2008).</li><li>Bacharier, L. B., Geha, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+IgE+regulation.">Molecular mechanisms of IgE regulation.</a> The Journal of allergy and clinical immunology. 105, 547-558 (2000).</li><li>Yokota, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+species+of+human+Fc+epsilon+receptor+II+(Fc+epsilon+RII/CD23):+tissue-specific+and+IL-4-specific+regulation+of+gene+expression.">Two species of human Fc epsilon receptor II (Fc epsilon RII/CD23): tissue-specific and IL-4-specific regulation of gene expression.</a> Cell. 55 (4), 611-618 (1988).</li><li>Boboila, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+end-joining+catalyzes+class+switch+recombination+in+the+absence+of+both+Ku70+and+DNA+ligase+4.">Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4.</a> The Journal of experimental medicine. 207, 417-427 (2010).</li><li>Nimmerjahn, F., Ravetch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fcgamma+receptors+as+regulators+of+immune+responses.">Fcgamma receptors as regulators of immune responses.</a> Nature reviews. Immunology. 8 (1), 34-47 (2008).</li><li>Callen, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=53BP1+mediates+productive+and+mutagenic+DNA+repair+through+distinct+phosphoprotein+interactions.">53BP1 mediates productive and mutagenic DNA repair through distinct phosphoprotein interactions.</a> Cell. 153 (6), 153-1280 (2013).</li><li>Wesemann, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+IgH+isotype-switched+B+cells+to+functional-grade+induced+pluripotent+stem+cells.">Reprogramming IgH isotype-switched B cells to functional-grade induced pluripotent stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (34), 13745-13750 (2012).</li><li>Wesemann, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immature+B+cells+preferentially+switch+to+IgE+with+increased+direct+S&#181;+to+S&#949;+recombination.">Immature B cells preferentially switch to IgE with increased direct S&#181; to S&#949; recombination.</a> The Journal of experimental medicine. 208 (13), 2733-2746 (2011).</li><li>Zarrin, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evolutionarily+conserved+target+motif+for+immunoglobulin+class-switch+recombination.">An evolutionarily conserved target motif for immunoglobulin class-switch recombination.</a> Nature immunology. 5, 1275-1281 (2004).</li><li>Fan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+class-specific+polyclonal+antibody-based+indirect+competitive+ELISA+for+detecting+fluoroquinolone+residues+in+milk.">Development of a class-specific polyclonal antibody-based indirect competitive ELISA for detecting fluoroquinolone residues in milk.</a> Journal of Zhejiang University. Science. B. 13 (7), 545-554 (2012).</li><li>Perez, O., Krutzik, P., Nolan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+kinase+signaling+cascades.">Flow cytometric analysis of kinase signaling cascades.</a> Methods in molecular biology (Clifton, N.J.). 263, 67-94 (2004).</li><li>Jamur, M., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fixatives+for+immunostaining.">Cell fixatives for immunostaining.</a> Methods in molecular biology (Clifton, N.J.). 588, 55-61 (2010).</li><li>Ishizaka, T., Ishizaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+passive+sensitization.+IV.+Dissociation+of+IgE+molecules+from+basophil+receptors+at+acid+pH.">Mechanisms of passive sensitization. IV. Dissociation of IgE molecules from basophil receptors at acid pH.</a> Journal of immunology (Baltimore, MD. : 1950). 112 (3), 1078-1084 (1950).</li><li>Erazo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+Maturation+Program+of+the+IgE+Response+In+Vivo.">Unique Maturation Program of the IgE Response In Vivo.</a> Immunity. 26, 191-203 (2007).</li><li>Xiong, H., Dolpady, J., Wabl, M., Curotto de Lafaille, M. A., Lafaille, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+class+switching+is+required+for+the+generation+of+high+affinity+IgE+antibodies.">Sequential class switching is required for the generation of high affinity IgE antibodies.</a> Journal of Experimental Medicine. 209, 353-364 (2012).</li><li>Yang, Z., Sullivan, B., Allen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+in+vivo+detection+reveals+that+IgE(+)+B+cells+are+restrained+by+an+intrinsic+cell+fate+predisposition.">Fluorescent in vivo detection reveals that IgE(+) B cells are restrained by an intrinsic cell fate predisposition.</a> Immunity. 36 (5), 857-872 (2012).</li><li>Berkowska, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+IgE(+)+B+cells+are+derived+from+T+cell-dependent+and+T+cell-independent+pathways.">Human IgE(+) B cells are derived from T cell-dependent and T cell-independent pathways.</a> The Journal of allergy and clinical immunology. , (2014).</li><li>He, J. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distinctive+germinal+center+phase+of+IgE++B+lymphocytes+limits+their+contribution+to+the+classical+memory+response.">The distinctive germinal center phase of IgE+ B lymphocytes limits their contribution to the classical memory response.</a> The Journal of experimental medicine. 210 (12), 2755-2771 (2013).</li><li>Keegan, A., Fratazzi, C., Shopes, B., Baird, B., Conrad, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+new+rat+anti-mouse+IgE+monoclonals+and+their+use+along+with+chimeric+IgE+to+further+define+the+site+that+interacts+with+Fc+epsilon+RII+and+Fc+epsilon+RI.">Characterization of new rat anti-mouse IgE monoclonals and their use along with chimeric IgE to further define the site that interacts with Fc epsilon RII and Fc epsilon RI.</a> Molecular immunology. 28 (10), 1149-1154 (1991).</li></ol>

The authors have no disclosures.

Anti-CD40 ve IL-4 ile kültürde fare dalak B hücreleri uyarımı IgG1 ve IgE 5, T yardımcı tip 2 (Th2) etkileşimi teşvik edici sınıf değiştirme simule eder. B hücreleri, toplam splenositler 12 ya da saflaştırılmış ve splenik B hücrelerinin 11 bağlamında CSR etkinleştirilebilir. Protokol (adım 2.3) de belirtildiği gibi, B hücresi zenginleştirme isteğe bağlıdır, ve yararlı olurdu olmadığını belirlemek için deneyci takdirine bağlıdır. CD45R (B...

Fare ve insan immünoglobulin ağır zinciri (IgH) sınıf anahtarı rekombinasyon (CSR) sırasında, IgH, sabit bölge ekzon (Cμ) silindi ve aşağı doğru sabit bölge eksonların çeşitli kümelerinin bir (Cı lH ler) (örneğin, Cγ değiştirilir μ Ig sınıflarına (örneğin, IgG, IgE ya da IgA) üretimine IgM üretimi ile ilgili bir anahtar elde Cε ve Ca&#39;sına). CSR CH s, 1 her bir grubu için 5 &#39;bölgesinde yer 1-10 kb sekansları anahtarı (s) bölgeleri içinde gerçekleşir. Aktivasyon Bağlı Sıtıdın Deaminaz (AID) enzim sitidinin deaminasyon etkinliği yoluyla CSR başlatır. IgE allerjik hastalık 2 anahtar arabulucu ve IgE üretilen ve atopik hastalığı için yeni tedavi yaklaşımları kapılarını açabilir düzenlenir nasıl büyük bir anlayıştır. Farelerde ve insanlarda olarak IgE, en sıkı bir şekilde düzenlenmiş Ig izotiptir. IgE normal tim düzeyleri binlerce tespit edilires diğer IgH daha az 3 isotypes, ama çok hastalık durumlarında 4 yükselmiş olabilir. Bununla birlikte, IgE CSR tam olarak anlaşılamamıştır. B hücrelerinin in vitro aktivasyonu, IL-4, anti-CD40 veya LPS ile kombinasyon halinde her iki IgG1 ve IgE 5 CSR indükler kullanılmıştır. Aktive B hücreleri çözünebilir IgE CSR sonra kültür salgılanan bağlayan düşük afiniteli IgE reseptörü FcεRII / CD23 2,6, ifade eder. Akış sitometrisi ile analiz, bu nedenle, reseptör bağlı IgE lekesi ile B hücreleri benzer endojen IgE 7 sentezleyen B hücreleri için. O fare B biliniyor olsa da hücreler IgG1 sınıf anahtarlama ölçümü ile müdahale görünmüyor deneyimlerimiz düşük afinite IgG reseptörü FcγRIIB1 (CD32) 8, ifade eder. Kültür içinde, B hücresi aktivasyonu sonrası IgE geçiş ölçüm Ancak, spesifik olmayan yüzeyine bağlı IgE analizi gizleyebilir. Yaygın boyama yöntemleriyle olmayan IgE ifade eden hücreler IgE için pozitif leke. Burada özetlenen fareler 9 arası gerçek IgE ifade B hücreleri KSS tahlilleri yapmak ve tespit etmek için kullanılmıştır bir stratejidir - 11. Tripsin ile aktive B hücrelerinin tedavisi, ortak bir laboratuar reaktif protein sindirimi, cytophylic ve membran hem de bağlı yüzey Ig kaldırır. Sitoplazmik IgE için sonraki permeabilization ve boyama, böylece gerçek IgE üreten hücreleri ortaya koymaktadır.

<table border="0" cellpadding="0" cellspacing="0" width="958">
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:301px;">Name</td>
			<td style="width:179px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:343px;">Comments</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-Mouse IgE FITC</td>
			<td>BD Pharmingen</td>
			<td>553415</td>
			<td>clone R35-72</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Rat Anti-Mouse IgG1 PE</td>
			<td>BD Pharmingen</td>
			<td>550083</td>
			<td>clone A85-1</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Methanol</td>
			<td>BDH</td>
			<td>BDH1135-4LP</td>
			<td>Keep at -20 &#176;C</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Falcon Cell Strainer 40 &#181;m Nylon</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Falcon Cell Strainer 70 &#181;m Nylon</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-Hu/Mo CD45R(B220) PerCP-Cy5.5</td>
			<td>eBioscience</td>
			<td>45-4052-82</td>
			<td>clone RA3-6B2</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">anti-Mouse/Rat CD40</td>
			<td>eBioscience</td>
			<td>16-0402-85</td>
			<td>clone HM40-3</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">RPMI Medium 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MEM-NEAA (100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Phosphate Buffered Saline (10X)</td>
			<td>Lifetechnologies (Corning)</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:67px;">MACS CD45R (B220) microbeads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-501</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MACS purification column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">IL-4</td>
			<td>PeproTech</td>
			<td>214-14</td>
			<td>Reconstitute in water or 0.1% BSA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Formalin Solution, Neutral Buffered, 10%</td>
			<td>Sigma Aldrich</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Trypsin-EDTA Solution (10X)</td>
			<td>Sigma Aldrich</td>
			<td>T4174-100mL</td>
			<td>5.0g Trypsin, 2.0g EDTA&#376;4Na per Liter of 0.9% NaCl</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Penicillin-Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>P0781-100mL</td>
			<td>10,000U Penicillin; 10 mg/mL Streptomycin (100X)</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">L-Glutamine 200mM</td>
			<td>Sigma Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">2-mercaptoethanol (99%)</td>
			<td>Sigma Aldrich</td>
			<td>M6250-100mL</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Red cell lysis buffer</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Rat Anti-Mouse IgM RPE/cy7</td>
			<td>SouthernBiotech</td>
			<td>1140-17</td>
			<td>clone 1B4B1</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">HyClone Fetal Calf Serum</td>
			<td>Thermo</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="161">
			<td height="161" style="height:161px;">B cell Stimulation media</td>
			<td></td>
			<td></td>
			<td style="width:343px;">B cell stimulation media consists of RPMI with fetal calf serum (15%), 20 mM HEPES, 1X MEM-NEAA, 2.0mM L-glutamine,&#160; 1X Penicillin-Streptomycin (Penicillin:100 U/ml, Streptomycin: 100 &#181;g/ml), Beta-mercaptoethanol (7 &#181;L/L), IL-4 (20 ng/mL), and anti-CD40 (1 &#181;g/mL)</td>
		</tr>
	</tbody>
</table>

detection of true ige-expressing mouse b lineage cells

B lenfosit immünoglobulin ağır zinciri (IgH) sınıf anahtarı rekombinasyon (CSR) ilk olarak, IgM IgA, IgE ve IgG olarak diğer IgH izotiplerinden geçer ifade edildiği bir süreçtir. In vitro IgH CSR ölçümü, moleküler ve hücresel immünoloji yönlerine DNA yeniden birleştirmesinden ve onarım kadar biyolojik süreçlerin bir dizi çalışma için bir temel yöntem. In vitro CSR deney aktive B hücreleri üzerindeki akış sitometrik ölçüm yüzey Ig ifade içerir. IgA ve IgG alt sınıflarının ölçümü kolay olmakla birlikte, bu yöntemle IgE ölçümü, çözünür IgE FcεRII bağlanma nedeniyle sorunludur / CD23 aktive B hücrelerinin yüzeyi üzerinde ifade edilmiştir. Burada kültür CSR geçirmiş fare B hücreleri, IgE üreten doğru bir şekilde ölçülmesi için benzersiz bir işlem tarif eder. yöntem cy ile sağlanan B tipi hücrelerin IgE üreten tespiti için izin hücre yüzeyleri üzerindeki IgE-CD23 komplekslerinin tripsin-sebepli bölünme dayanırtoplasmic lekelenmesi. Bu prosedür IgE KSS akış sitometri analizi için uygun bir çözüm sunuyor.

Bu prosedür başarıyla fare B hücrelerinde IgE CSR çalışma uygulamaya konmuştur. Daha önce tarif edildiği gibi 11 CSR ölçüm etkinliği tespit etmek için, anti-CD40 ve IL-4 ile birlikte fare dalak B hücreleri stimüle. Stimülasyon beş gün sonra, hücreler toplandı ve protokol floresan-etiketli IgM, IgG1, IgE ve B220 (CD45R) antikorlar ile, yukarıda tarif edilen ve lekeli kullanılarak işlenmiştir. (Şekil 1), IgE + hücrelerin net bir nüfusu temiz tripsin tedavi (...

Watch this Scientific Journal Video about Detection of True IgE-expressing Mouse B Lineage Cells at JoVE.com

Detection of True IgE-expressing Mouse B Lineage Cells

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 ...

detection-of-true-ige-expressing-mouse-b-lineage-cells

Research

JoVE Journal

Immunology and Infection

10.9K Görüntüleme.  Brigham and Women's Hospital and Harvard Medical School. 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.

Video: Detection of True IgE-expressing Mouse B Lineage Cells

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

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 ...

İmmünoloji ve Enfeksiyon

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 ...

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 ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

PNA-FISH ile Fare B Lenfositlerindeki kromozom anormallikleri hızlı Analizi

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

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, ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Hızlı moleküler algılama ve Nezle Virüsleri A ve B farklılaşması

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

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 ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasome harekete geçirmek ve Pyroptotic hücre ölümü fare kemik iliği türevi makrofajlar içinde tespiti

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Kimlik ve yalıtım Oligopotent ve Lineage için kabul edilen miyeloid ataları fare kemik iliği dan

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

Algılama düşük kopya sayısı Vitro hepatit B enfeksiyonu tarafından kurulan entegre Viral DNA

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...