This work was supported by the Swedish Cancer Society (11 0253, 13 0469), the Swedish Research Council (K2012-99X-21959-01-3), Marcus Borgstr&#246;ms Foundation and the Swedish Research Council through a grant to the Uppsala RNA Research Centre (2006-5038-36531-16). We are indebted to the BioVis core facility at Uppsala University for much help with the FACS analysis.

该协议描述了从人类患者材料淋巴细胞的分离，因此，需要一个伦理批准。在本研究中所做的工作给予的乌普萨拉伦理审查委员会（DNR。三百八十七分之二千〇一十三）。 1.分离单个核人力腭扁桃体细胞（MNCs）的警告:人类起源如血液，组织或体液的所有非屏蔽材料应被视为可能受感染的材料。因此，处理的人体组织推荐的生物安全的做法应该得到遵守。 注意:为了避免污染，所有的解决方案和细胞培养设备必须是无菌的。所有缓冲液和溶液应预先冷却并保持在冰上。扁桃体组织分离出的细胞应保持在冰上处理。 <ol><li>获得从接受扁桃体切除术或扁桃体部分切除术（图2）的患者新鲜扁桃体。急跌组织clum的ps到装有冰冷无菌的Hanks平衡盐溶液（HBSS）在50毫升离心管中补充有5％胎牛血清（FBS），10mM的谷氨酰胺，0.05毫克/毫升庆大霉素和1％抗菌 - 抗霉菌混合物（青霉素，链霉素和两性霉素B）。 </li><li>保持与淹没扁桃体组织样品管在任何时候都在冰上。尝试尽快处理的扁桃体，并最迟手术切除后3小时。 </li><li>广场上冰60毫米的塑料细胞培养板的扁桃体和保持蘸有HBSS组织。删除可见血栓，脂肪和结缔组织从扁桃体表面干净的镊子。 </li><li>使用含有5ml HBSS中一个新的60毫米细胞培养板。切割扁桃体组织丛成使用无菌​​剪刀和/或手术刀的3-10毫米的碎片。 </li><li>制备装有10ml HBSS的新60毫米的细胞培养板，并放置一个100μm的塑料的细胞过滤的HBSS溶液。 </li><li>转移迪斯ected扁桃体碎片进入细胞过滤用无菌镊子。使用塑料注射器的柱塞端，顺利地通过细胞过滤器挤压组织碎片。确保扁桃体片段完全沉浸在HBSS中。 </li><li>丢弃细胞过滤与组织遗体和从60毫米细胞培养板转移得到的细胞悬液至50ml塑料离心管中。留在冰上的管继续进行步骤1.8和1.9，而。 </li><li>在大扁桃体的情况下，大于2厘米大小（图2），挤压扁桃体片段在两个细胞滤网，以避免在过滤器网眼堵塞。 </li><li>获得35毫升细胞悬浮液的最终体积。 注意 :在这一步，可以制备细胞悬浮液为低温保存（参见部分2）。 </li><li>加入10毫升如Ficoll密度梯度溶液例如到一个新的50ml离心管中。轻轻地覆盖在细胞suspens离子上的密度梯度溶液的顶部（步骤1.9）。避免了细胞悬浮液和密度梯度溶液的混合。 注意 :密度梯度溶液必须被升温至室温。 </li><li>离心细胞悬浮液在水平转子在700×g离心20分钟，在室温。不要激活离心机快制动可能会破坏梯度的制动功能。此外，使用可在离心机的最低加速功能。 注意 :在此之后的离心步骤，观察跨国公司，为蓬松的白色层在界面处，和红血细胞（红细胞），成纤维细胞和细胞碎片的沉淀物在管的底部。 </li><li>小心地用10毫升移液管收集跨国公司层。将细胞悬浮液到一个新的50ml离心管中。 </li><li>加25毫升冰冷PBS，以细胞悬浮液和旋管中，在300×g离心5分钟以水平转子在4℃下。 </li><li>用25毫升移液管除去上清液并重复细胞沉淀洗涤步骤两次以上。最后重悬细胞沉淀在15ml PBS中。 注意 :最后的洗涤步骤之后，就可以冷冻保存的细胞悬浮液（参见2）。 </li><li>申请10微升细胞悬液到血球细胞计数室和计数在显微镜下的细胞数。分配细胞的适量（例如，2-3×10 7个）的15ml离心管中用于随后磁珠分离。保持这个等分在冰上，直到步骤3.2。 </li></ol> 2.冷冻扁桃体细胞<ol><li>制备冷冻介质（90％FBS和10％DMSO）中，并保持它在冰上。对于长期储存保持冷冻介质在-20℃。 </li><li>确定使用血球细胞计数室（步骤1.15）细胞的总数。根据所需的冷冻的细胞密度计算所需冷冻介质的量（例如，10 7细胞个冰冻培养基/毫升）。 </li><li>离心细胞悬浮液在300×g离心5分钟。滗析出上清液，而不干扰细胞沉淀重悬在冰冷的冷冻介质（来自步骤2.1）将细胞沉淀。 </li><li>分配1ml等分细胞悬浮液成设计用于长期储存在液氮中无菌小瓶。冻结小瓶中的异丙醇室，并将其存储在-80°CO / N。对于长期保存的小瓶转移到含有贮槽或-140℃的细胞冷冻液氮。 </li><li>解冻小瓶冷冻细胞，迅速温暖他们在37℃水浴。解冻时立即，分散细胞悬浮到10ml预温热的HBSS（用补充剂，步骤1.1）在15ml离心管中。旋管中于300×g离心5分钟，除去HBSS和重悬细胞沉淀在于HBSS所需的细胞密度（与补充剂，步骤1.1）。 注:跨国公司房颤的可行性之三解冻是很重要的，因为死细胞的存在将降低纯化的B和T淋巴细胞的最终产率。 注意 :根据我们的经验细胞存活率小于80％会降低细胞分离效率。 </li></ol> 3.积极选择T淋巴细胞人口从扁桃体跨国公司 注 1:该协议是基于正选择从使用耦合到CD3抗体磁珠扁桃体跨国公司人CD3 + T淋巴细胞的。有可能从新鲜（第1）或冷冻（第2节）跨国公司开始本节。 注2:开始用3×10 7跨国公司。不要超过这个手机号码，因为分离柱可能阻塞，这将降低隔离效率。使用大柱，如果更多的细胞将被处理。在这个协议中使用的量已经为我们的细胞的数量被实验优化海关在我们的实验装置。 <ol><li>制备分离缓冲液[PBS（pH为7.2），0.5％牛血清白蛋白（BSA）和2mM EDTA〕。通过0.45微米的过滤器，并储存在4℃下过滤分离缓冲液。 </li><li>旋转跨国公司悬架（步骤1.14），在300 XG 5分钟，在4℃。重悬的冰冷分离液在240微升所得细胞沉淀（每10 7个细胞80微升）。细胞悬液转移到无菌的2毫升管。 </li><li>加入20微升的CD3磁性抗体与细胞溶液。孵育管1小时，在4℃下用连续轻柔混合，以保持细胞在悬浮液中。 </li><li>传输所有的细胞悬液入15ml试管含有5ml冰冷分离缓冲液洗涤细胞。 </li><li>离心管中，在300×g离心10分钟，在4℃下，在水平转子。 </li><li>同时成立了磁选柱。附加磁性分离到支架和放置列在组合通道员。通过应用500微升冰冷的分离缓冲液洗柱。丢弃通过流。 </li><li>放置柱下方的新款15 ml收集管。保持在冰上收集管。 </li><li>用移液管弃去上清液（步骤3.5）和轻轻悬浮于500μl分离缓冲液的细胞沉淀。应用细胞悬液的预洗塔的顶部，并让它运行通过。 注:细胞团块会堵塞柱，从而降低流速。为了防止出现这种问题，将在塔的顶部的40微米的塑料细胞过滤。通过这个网格传递细胞将大大提高了该方法的效率。 </li><li>用1.5ml的分离缓冲液（500微升，10 7个细胞 ）洗涤塔4倍。等待列储到是空的（没有液体应在塔中观察到的）施加下一洗涤步骤之前进行。 注意:获取CD3未标记细胞，视为乙淋巴细胞部分，因为它们是洗脱到收集管。 </li><li>拆下分离器和地点列到新款15 ml收集管。移液管2毫升分离缓冲液上柱。使用与列提供的柱塞洗脱阳性选择T淋巴细胞，这被认为是T淋巴细胞组分。 </li><li>确定纯化的细胞（步骤1.15），如果必要的数目。细胞悬液现在已经准备好为下游实验。 </li></ol>孤立4.流式细胞仪分析扁桃体B和T淋巴细胞注意:多聚甲醛溶液有刺激性，并可能致癌。穿戴合适的防护服，手套和防护眼镜/面罩。 注1:该协议描述通过荧光活化细胞的方法中的分离的B和T细胞的直接染色分选（FACS）分析。这个步骤的主要目的是评估所述分离的细胞群自动对焦的纯度之三CD3磁性抗体分离。为此建立B和T细胞标记物，荧光团共轭的单克隆抗体CD20-FITC和CD2-APC，被使用。还列入同种型对照抗体的强烈建议区分非特异性"背景"的CD2和CD20抗体（见步骤4.8）的结合。 注2:有可能保持在1％多聚甲醛的纯化的细胞级分在黑暗中（PFA）溶液，在4℃，直至染色的时间 （例如，第二天。）。也同样地进行适用，如果有染色和FACS分析之间的时间间隔。请记住，在这两种情况下的细胞应该正确地与（含0.2％BSA的PBS）缓冲液，对染色或FACS分析之前PBSA洗涤。 <ol><li>取出从步骤1.15跨国公司6×10 6个细胞（前施加到列）从步骤3.10从步骤3.9和T淋巴细胞部分，B淋巴细胞部分。 </li><li>旋转细胞使用水平转子悬浮在300 XG 5分钟，在4℃。 </li><li>用移液管除去上清液并用1ml冰冷的PBSA洗细胞沉淀一次。旋转细胞悬浮液在300×g离心5分钟，在4℃。用吸管去除上清，重悬 细胞在600微升冰冷PBSA缓冲液中。 </li><li>加入100微升细胞悬浮液，以在FACS管的底部。 </li><li>加入100微升含10％热灭活人血清，每管的PBS，拌匀孵育在室温〜1分钟或20分钟冰。 注:B淋巴细胞携带Fc受体。为了阻断Fc受体的纯化的细胞级分一起温育10％热灭活的人血清。的人血清是热在56℃下失活温育1小时。划分热灭活血清分成小等分试样并储存在-20℃。 </li><li>离心细胞悬浮液在300×g离心5分钟，在4℃下，在SW荷兰国际集团出转子。 </li><li>用移液管除去上清液并用1ml PBSA洗涤细胞沉淀。重复离心（300×g离心5分钟，4℃）。 </li><li>加入100微升PBSA到冰上每管。添加荧光团共轭单克隆抗体的合适量，根据制造商的建议（例如，20微升抗CD20和/或5微升抗CD2抗体在100μlPBSA的）。注意:不要忘记分配足够的对照管的流式细胞仪分析。在这个协议中的下列控制应包括:1）染色单独的抗-CD2和抗CD20抗体的细胞，2）染色的细胞与单独的抗-CD2和抗CD20同种型对照抗体和，3）细胞不加的抗体。 </li><li>短暂涡旋振荡试管并在黑暗中温育30分钟，在4℃。 </li><li>与PBSA洗2次。加入2毫升1％PFA溶液的样品。让细胞保留在煤灰溶液在476下在黑暗直到FACS分析的时间。 </li><li>立即运行样本在FACS仪器之前，（在4℃下300×g离心5分钟）用PBSA洗细胞2次。在1ml PBS中重悬样品保持在4℃（或在冰上），避光，之前在流式细胞仪的分离。 </li><li>做下面的流式细胞仪制造商提供的流量协议的FACS分类。 </li></ol>在隔离扁桃体B和T淋巴细胞5 PCR检测腺病毒的DNA 注1:腺病毒六邻体基因的引物是AdRJC1（5&#39;-GACATGACTTTCGAGGTCGATCCCATGGA-3&#39;）和AdRJC2（5&#39;-CCGGCTGAGAAGGGTGTGCGCAGGTA-3&#39;）11。这些引物产生的139 bp的扩增。主机18S rRNA基因可以与引物tp206（5&#39;-CCCCTCGATGCTCTTAGCTG-3&#39;）和TP207（5&#39;-TCGTCTTCGAACCTCCGACT-3&#39;）进行检测。预期的扩增子大小为300基点。 注2:每个PCR运行包括阴性对照（蒸馏水H 2 O）和从人B细胞系（BJAB）得到的阳性DNA对照感染人类腺病毒5型12。 <ol><li>提取的DNA（来自至少1×10 6个细胞，步骤3.11），使用氧化硅膜基于柱纯化方法。采用苯酚和异硫氰酸胍溶液 13中提取RNA。 </li><li>添加从B和T淋巴细胞100 ng的DNA至含有1x HF缓冲液，0.2mM的的三磷酸脱氧核苷酸的混合物（dNTP），0.25μM的每种引物和高保真DNA聚合酶在20微升的总体积1 U的反应混合物。 </li><li>以下循环条件下进行PCR扩增:30秒变性，在98℃下，然后是98℃30个循环持续10秒，67℃（六邻体）或58℃（18S rRNA）的30秒和72℃ 30秒。 PCR反应结束时的7分钟，在72℃最终延伸步骤。 </li><li>分离得到的PCR amplif上于1×TBE 1.5％琼脂糖凝胶ication产品缓冲液和通过用核酸染料染色可视化的预期条带。 </li></ol>

<ol>
	<li>Nave, H., Gebert, A., Pabst, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+immunology+of+the+human+palatine+tonsil.">Morphology and immunology of the human palatine tonsil.</a> Anat Embryol (Berl). 204, 367-373 (2001).</li><li>Perry, M., Whyte, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+of+the+tonsils.">Immunology of the tonsils.</a> Immunology today. 19, 414-421 (1998).</li><li>Alkhalaf, M. A., Guiver, M., Cooper, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+quantitation+of+adenovirus+DNA+from+human+tonsil+and+adenoid+tissues.">Prevalence and quantitation of adenovirus DNA from human tonsil and adenoid tissues.</a> J Med Virol. 85, 1947-1954 (2013).</li><li>Brandtzaeg, P., Halstensen, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+and+immunopathology+of+tonsils.">Immunology and immunopathology of tonsils.</a> Adv Otorhinolaryngol. 47, 64-75 (1992).</li><li>Imai, 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=Epstein-Barr+virus+(EBV)-carrying+and+-expressing+T-cell+lines+established+from+severe+chronic+active+EBV+infection.">Epstein-Barr virus (EBV)-carrying and -expressing T-cell lines established from severe chronic active EBV infection.</a> Blood. 87, 1446-1457 (1996).</li><li>Veen, J., Lambriex, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+adenovirus+to+lymphocytes+in+naturally+infected+human+tonsils+and+adenoids.">Relationship of adenovirus to lymphocytes in naturally infected human tonsils and adenoids.</a> Infect Immun. 7, 604-609 (1973).</li><li>Friedman, M., Wilson, M., Lin, H. C., Chang, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Updated+systematic+review+of+tonsillectomy+and+adenoidectomy+for+treatment+of+pediatric+obstructive+sleep+apnea/hypopnea+syndrome.">Updated systematic review of tonsillectomy and adenoidectomy for treatment of pediatric obstructive sleep apnea/hypopnea syndrome.</a> Otolaryngol Head Neck Surg. 140, 800-808 (2009).</li><li>Garnett, C. T., 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=Latent+species+C+adenoviruses+in+human+tonsil+tissues.">Latent species C adenoviruses in human tonsil tissues.</a> J Virol. 83, 2417-2428 (2009).</li><li>Garnett, C. T., Erdman, D., Xu, W., Gooding, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+quantitation+of+species+C+adenovirus+DNA+in+human+mucosal+lymphocytes.">Prevalence and quantitation of species C adenovirus DNA in human mucosal lymphocytes.</a> J Virol. 76, 10608-10616 (2002).</li><li>Perez, M. E., Billordo, L. A., Baz, P., Fainboim, L., Arana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+memory+B+cells+isolated+from+blood+and+tonsils+are+functionally+distinctive.">Human memory B cells isolated from blood and tonsils are functionally distinctive.</a> Immunol Cell Biol. 92, 882-887 (2014).</li><li>Cooper, R. J., Yeo, A. C., Bailey, A. S., Tullo, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+polymerase+chain+reaction+assay+for+rapid+diagnosis+of+conjunctivitis.">Adenovirus polymerase chain reaction assay for rapid diagnosis of conjunctivitis.</a> Invest Ophthalmol Vis Sci. 40, 90-95 (1999).</li><li>Zhang, Y., Huang, W., Ornelles, D. A., Gooding, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+adenovirus+latency+in+human+lymphocyte+cell+lines.">Modeling adenovirus latency in human lymphocyte cell lines.</a> J Virol. 84, 8799-8810 (2010).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction.">Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.</a> Anal. biochem. 162, 156-159 (1987).</li><li>Watanabe, T., Yoshizaki, K., Yagura, T., Yamamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+antibody+formation+by+human+tonsil+lymphocytes.">In vitro antibody formation by human tonsil lymphocytes.</a> J Immunol. 113, 608-616 (1974).</li></ol>

The authors have nothing to disclose.

一个影响该协议的结果的最重要因素是使用新鲜扁桃体材料作为原料。因此，扁桃体样品应在3个小时的手术后处理。扁桃体可以从成人和儿童中获得。从孩子扁桃体材料通常是较小的由于局部手术切除扁桃体（扁桃体部分切除术）的。因此，从扁桃体部分切除术的样品（1×10 7 -1×10 8）中得到跨国公司的数目较少相比扁桃体样品（1×10 8 -2×10 9）。然而，细胞分离?...

Tonsils are collections of incompletely encapsulated lymphoid tissues that lie under, and in contact with, the epithelium in the upper aero-digestive tract. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. The paired palatine tonsils together with the nasopharyngeal tonsil (adenoid), paired tubal tonsils and lingual tonsils constitute the so-called &#8220;Waldeyer&#180;s ring&#8221;. The latter is responsible for the initial contact between inhaled or ingested pathogens and the lymphoid tissues of the aerodigestive tract1,2. Indeed, numerous reports have shown that both bacterial and viral antigens can be detected in palatine tonsil tissue samples2-6.
The palatine tonsils are composed of dense lymphoid tissue covered by a stratified squamous non-keratinising epithelium. The tonsils have numerous crypts, epithelial invaginations, which penetrate the parenchyma increasing the surface area. Histologically, the palatine tonsils contain numerous lymphoid follicles with germinal centers, which are the sites for B cell maturation and differentiation (B-cell areas). Likewise, the palatine tonsils encompass T cells, which are mainly located in the extrafollicular regions (T-cell areas). In addition to the B and T cells, also various follicular dendritic cells can be detected in palatine tonsils1,2. 
Due to their anatomic location, the palatine tonsils are easily accessible by surgical interventions. For example, surgical removal of tonsils, known as tonsillectomy, is routinely carried out worldwide7. In children with tonsillar hyperplasia, a partial surgical removal of the tonsils (tonsillotomy) is sometimes used, causing less postoperative pain to the patients. Considering the accumulation of various pathogens in tonsils, surgically removed tonsils provide a unique opportunity to study the influence of viral and bacterial agents on tonsillar lymphocyte functions2,8. Furthermore it is possible to study if some pathogens prefer to reside in specific cell subpopulations9. In addition, as the tonsils are rich source of B lymphocytes, isolated tonsillar B lymphocytes can be efficiently used to study the activity of different B cell subpopulations10. However, as the palatine tonsils contain a mixture of cell types an efficient method to separate the different cell subpopulations is needed. 
Here, we describe a simple method for efficient and rapid isolation of tonsillar B and T cell populations from human palatine tonsils by using a magnetic-activated cell separation technique (Figure 1). The method described here is useful for scientists who want to assess the role of different infectious agents in human lymphoid organs such as palatine tonsils.

<table><tbody><tr><td>Hanks balanced salt solution (HBSS)&amp;nbsp;</td><td>Gibco</td><td>14175-053</td><td>Contains 5% fetal bovine serum (FBS), 10 mM Glutamine,&amp;nbsp; &amp;nbsp;0.05 mg/ml Gentamicin and 1% Antibiotic-Antimycotic mix</td></tr><tr><td>Freezing medium</td><td></td><td></td><td>90% FBS and 10% DMSO</td></tr><tr><td>MACS buffer</td><td></td><td></td><td>PBS (pH 7.2), 0.5% BSA and 2mM EDTA</td></tr><tr><td>PBSA&amp;nbsp;</td><td></td><td></td><td>PBS containing 0.2% BSA</td></tr><tr><td>PFA</td><td></td><td></td><td>PBS containing 1% paraformaldehyde (PFA). Make fresh. PFA is suspected carcinogen. Wear gloves and goggles.</td></tr><tr><td>Antibiotic-Antimycotic mix</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>60 mm Petridish</td><td>Nunc</td><td>150326</td><td></td></tr><tr><td>Dissecting foreceps</td><td>Fisher Scientific</td><td>1381241</td><td></td></tr><tr><td>Straight iris scissors&amp;nbsp;</td><td>Fisher Scientific</td><td>12912055</td><td></td></tr><tr><td>disposable scalpels</td><td>Swann-Morton</td><td>REF 0501</td><td></td></tr><tr><td>100 &amp;mu;m plastic cell strainer</td><td>Corning Life Sciences</td><td>352360</td><td></td></tr><tr><td>40 &amp;mu;m plastic cell strainers&amp;nbsp;</td><td>Corning Life Sciences</td><td>352340</td><td></td></tr><tr><td>2 ml plastic syringe</td><td>BD Biosciences&amp;nbsp;</td><td>300185</td><td></td></tr><tr><td>15 ml conical centrifuge tubes</td><td>SARSTEDT</td><td>62554502</td><td></td></tr><tr><td>50 ml conical centrifuge tubes</td><td>SARSTEDT</td><td>62547254</td><td></td></tr><tr><td>Low-speed centrifuge with fixed-angle or swinging-bucket rotor</td><td>Thermo Scientific Heraeus Megafuge 16R</td><td></td><td></td></tr><tr><td>Ficoll&amp;ndash;Hypaque</td><td>Sigma-Aldrich</td><td>F5415-50ML</td><td>Ficoll solution</td></tr><tr><td>Fetal calf serum</td><td>Biological industries</td><td>040071A</td><td></td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>SIGMA</td><td>D2650-5X5ML</td><td></td></tr><tr><td>MACS MS columns&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>MACS human CD3 MicroBeads</td><td>Miltenyi Biotec</td><td>130-092-881</td><td></td></tr><tr><td>MACS separator (Octo MACS)</td><td>Miltenyi Biotec</td><td>130-042-109</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Merck Millipore</td><td>1120180100</td><td></td></tr><tr><td>EDTA</td><td>AnalaR NORMAPUR</td><td>20302.293</td><td></td></tr><tr><td>CD2 conjugated to allophycocyanin (CD2-APC)&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>560642</td><td></td></tr><tr><td>CD20 conjugated to fluorescein isothiocyanate (CD20-FITC)&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>556632</td><td></td></tr><tr><td>Human serum&amp;nbsp;</td><td>Rockland Immunochemicals</td><td>D119-0100</td><td></td></tr><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>Thermo Scientific</td><td>F-530L</td><td></td></tr><tr><td>FACS tubes</td><td>BD Falcon</td><td>352003</td><td></td></tr><tr><td>Cryotube</td><td>SARSTEDT</td><td>72379</td><td></td></tr><tr><td>BD LSRII flowcytometer</td><td>BD Biosciences&amp;nbsp;</td><td></td><td></td></tr><tr><td>BD FACSDiva 4.1 software</td><td>BD Biosciences&amp;nbsp;</td><td></td><td></td></tr><tr><td>Nucleospin Blood&amp;nbsp;</td><td>Macherey-Nagel</td><td>740951.50</td><td>DNA isolation kit</td></tr><tr><td>TRIzol&amp;nbsp; reagent</td><td>Life technologies</td><td>15596</td><td>RNA isolation reagent</td></tr><tr><td>Hemocytometer</td><td>The Paul Marienfeld GmbH &amp;amp; Co. KG</td><td>0610030</td><td>cell counter device</td></tr><tr><td>GelRed</td><td>Biotium</td><td>41003</td><td>Nucleic acid gel stain&amp;nbsp;</td></tr></tbody></table>

efficient isolation protocol for b and t lymphocytes from human palatine tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

的扁桃体跨国公司结果在B和T淋巴细胞的高度纯化的亚群有效分离。这是通过FACS分析使用抗CD20和抗-CD2抗体，以检测B和T淋巴细胞群，分别（图3A）证实。与此相反，跨国公司导致细胞级分是从两个B和T细胞亚群（图3B）细胞的混合物低效的分离。 分离的扁桃体B和T淋巴细胞（图3A）是足够的质量为下游分析样核酸隔离。在当前的协议，DNA和...

Watch this Scientific Journal Video about Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils at JoVE.com

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

从人类腭扁桃体B和T淋巴细胞有效隔离协议

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

efficient-isolation-protocol-for-b-t-lymphocytes-from-human-palatine

Research

Education

JoVE Core

JoVE Journal

Medical-Surgical Nursing

Immunology and Infection

Unit 1

Upper Respiratory Tract Infections

SCIENTISTS IN ACTION

15.7K 次观看.  Uppsala Biomedical Center, Uppsala University. Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

视频: Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer&#39;s patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes.
Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on&#160;TFH and GC B cell subsets.

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer&#39;s Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

从小鼠的补丁中提取体液免疫的关键参与者: 先进和优化的淋巴细胞分离协议

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense ...

科研

癌症研究

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides (MF) and S&#233;zary syndrome (SS) represent the most common subtypes. Despite a number of studies on characterizing gene expression, genetic alterations, and epigenetic abnormalities of CTCL, the molecular pathogenesis of MF/SS remains unclear. MF refers to the more common CTCL with a skin-predominance, and is usually limited to skin, whereas&#160;SS is an aggressive leukemic variant of CTCL with widespread skin involvement and is characterized by neoplastic distribution mainly involving blood, skin, and lymph node. Focusing on clinical practice, the identification of gene expression biomarkers has&#160;enormous potential to improve diagnosis and treatment of MF/SS. Indeed, recent transcriptomic studies have identified potential diagnostic biomarkers from differences in gene expression between normal and malignant T cells, which may improve our understanding of SS biology, and reveal potential therapeutic targets. This manuscript describes a detailed reproducible protocol for the isolation of peripheral blood mononuclear cells from fresh whole blood from patients diagnosed with SS, selection of CD4+ memory T cells (CD4+CD45RO+ T cells), chemical stimulation, and preparation of RNA suitable for transcriptomic profiling to discover novel prognostic molecular markers to gain additional insight in disease etiology. The stimulation using chemical agonist to activate nuclear regulation provides more specific assessment for pathways important in the dynamic transcription regulation and gene expression and eliminates confounding defects that may arise from upstream signaling defects arising from TCR antigen loss at the cell membrane. The data obtained from comparison of transcriptome of unstimulated to stimulated SS T cells unmasks functional regulatory gene expression defects not evident from analysis of quiescent unstimulated cells. Furthermore, the method outlined from this approach can be adapted for studying T cell gene expression defects in other T cell immune diseases.

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

从Sézary综合征患者中分离人外周血单核细胞和CD4 + T细胞以进行转录组学分析

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

免疫与感染

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

的分离，分化和人类的定量分泌抗体的B细胞从血液:酶联免疫斑点作为体液免疫的功能读出

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

分离及小鼠淋巴细胞的激活

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

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

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

小鼠小肠免疫系统淋巴细胞的分离

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

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

生物学

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

单核细胞分离研究人类粘膜淋巴组织中先天免疫反应

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

Isolation and Purification of B Cells from Human Peripheral Blood

Immediately after obtaining a 10-milliliter blood sample from the median cubital vein into a 15-milliliter tube containing potassium-supplemented EDTA, invert the tube several times to prevent clot formation. Then, add 35 milliliters of autoclaved red blood cell lysis buffer to the cells for a 5-minute incubation at room temperature.
When light can be observed through the tube, centrifuge the blood and confirm the presence of a white pellet. Remove the residual lysis buffer with 10 milliliters of autoclave to PBS, and resuspend the pellet in 10 milliliters of RPMI 1640 medium.
Plate the cells in a 10-centimeter culture dish for a 30-minute incubation at 37 degrees Celsius and 5% carbon dioxide. Then, gently swirl the culture dish a few times and transfer the floating cells into a plastic 15-milliliter conical tube.
After two washes by centrifugation, resuspend the pellet at a 5 to 10 x 106 cells per milliliter concentration in 200 microliters of cold PBS buffer, and add 5 microliters of biotinylated anti-human antibody cocktail specific for blood cells per 1 x 106 cells.
After a 30-minute incubation on ice, wash the cells in a 10-fold excess volume of sterile PBS, and resuspend the pellet in 5 microliters of streptavidin-conjugated microbeads per 1 x 106 cells for a 30-minute incubation on ice. At the end of the incubation, add 2 milliliters of PBS buffer to the cells and place the tube onto a magnetic stand for 8 minutes at room temperature.
When the microbeads have attached to the wall of the tube, carefully transfer the supernatant into a new conical tube, and add 2 more milliliters of PBS buffer to the cells. After the second supernatant collection, collect the pooled cells by centrifugation, and transfer the cells to a 5-milliliter polystyrene tube in fresh PBS buffer at a 1 x 107 cells per milliliter concentration.