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>

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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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Hanks balanced salt solution (HBSS)&#160;</td>
			<td>Gibco</td>
			<td>14175-053</td>
			<td>Contains 5% fetal bovine serum (FBS), 10 mM Glutamine,&#160;</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>&#160;0.05 mg/ml Gentamicin and 1% Antibiotic-Antimycotic mix</td>
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		<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&#160;</td>
			<td></td>
			<td></td>
			<td>PBS containing 0.2% BSA</td>
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		<tr>
			<td>PFA</td>
			<td></td>
			<td></td>
			<td>PBS containing 1% paraformaldehyde (PFA). Make fresh.</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>PFA is suspected carcinogen. Wear gloves and goggles.</td>
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			<td></td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Antibiotic-Antimycotic mix</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
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		<tr>
			<td>100-mm petridish</td>
			<td>Nunc</td>
			<td>172958</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting foreceps</td>
			<td>Fisher Scientific</td>
			<td>1381241</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight iris scissors&#160;</td>
			<td>Fisher Scientific</td>
			<td>12912055</td>
			<td></td>
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		<tr>
			<td>disposable scalpels</td>
			<td>Swann-Morton</td>
			<td>REF 0501</td>
			<td></td>
		</tr>
		<tr>
			<td>100&#956;m plastic cell strainer</td>
			<td>Corning Life Sciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#956;m plastic cell strainers&#160;</td>
			<td>Corning Life Sciences</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>2-ml plastic syringe</td>
			<td>BD Biosciences&#160;</td>
			<td>300185</td>
			<td></td>
		</tr>
		<tr>
			<td>15-ml conical centrifuge tubes</td>
			<td>SARSTEDT</td>
			<td>62554502</td>
			<td></td>
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		<tr>
			<td>50-ml conical centrifuge tubes</td>
			<td>SARSTEDT</td>
			<td>62547254</td>
			<td></td>
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		<tr>
			<td>Low-speed centrifuge with fixed-angle or swinging-bucket rotor</td>
			<td colspan="2">Thermo Scientific Heraeus Megafuge 16R</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll&#8211;Hypaque</td>
			<td>Sigma-Aldrich</td>
			<td>F5415-50ML</td>
			<td>Ficoll solution</td>
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		<tr>
			<td>Fetal calf serum</td>
			<td>Biological industries</td>
			<td>040071A</td>
			<td></td>
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		<tr>
			<td>Dimethyl sulphoxide (DMSO)</td>
			<td>SIGMA</td>
			<td colspan="2">D2650-5X5ML</td>
		</tr>
		<tr>
			<td>MACS MS columns&#160;</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>
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		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Merck Millipore</td>
			<td>1120180100</td>
			<td></td>
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		<tr>
			<td>EDTA</td>
			<td>AnalaR NORMAPUR</td>
			<td>20302.293</td>
			<td></td>
		</tr>
		<tr>
			<td>CD2 conjugated to allophycocyanin (CD2-APC)&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>560642</td>
			<td></td>
		</tr>
		<tr>
			<td>CD20 conjugated to fluorescein isothiocyanate (CD20-FITC)&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>556632</td>
			<td></td>
		</tr>
		<tr>
			<td>Human serum&#160;</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&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva 4.1 software</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleospin Blood&#160;</td>
			<td>Macherey-Nagel</td>
			<td>740951.50</td>
			<td>DNA isolation kit</td>
		</tr>
		<tr>
			<td>TRIzol&#160; reagent</td>
			<td>Life technologies</td>
			<td>15596</td>
			<td>RNA isolation reagent</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>The Paul Marienfeld GmbH &#38; Co. KG</td>
			<td>0610030</td>
			<td>cell counter device</td>
		</tr>
		<tr>
			<td>GelRed</td>
			<td>Biotium</td>
			<td align="right">41003</td>
			<td>Nucleic acid gel stain&#160;</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

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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

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

免疫与感染

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

小鼠生殖道黏膜淋巴细胞分离

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

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&amp;#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 ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

使用Jsubtomo从电子Cryotomography重构平均病毒包膜糖蛋白穗

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

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.

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

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

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

人THP-1巨噬细胞通过核转高效转染

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

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.

从排序的永生B细胞产生的重组人IgG单克隆抗体

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

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

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

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

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