This study was supported by a research grant from the Ministry of Science and Technology of the Executive Yuan of Taiwan (NSC99-2320-B-002-011). I would like to acknowledge the excellent service provided by the Flow Cytometric Analyzing and Sorting Core of the First Core Laboratory in College of Medicine of National Taiwan University.

人体外周血必须从知情同意下健康供者获得，利用血液样本必须符合个别机构审查委员会制定的指导方针批准。在这项研究中，要使用的协议人体血液流式细胞术（ 图1）和酶联免疫斑点试验（ 图3）的结果的示威是经台大医院的内部审查委员会（协议号201307019RINB）。 1.分离人外周血B细胞的分离纯化<ol><li>绘制〜10毫升的血液从肘正中静脉（在肘窝前部到肘部）到含有K 2 EDTA（1.5至2.0毫克/毫升血）有15毫升管中并立即翻转试管几次，以防止凝块形成。 </li><li>加的高压灭菌（121℃，15分钟）的红血细胞（RBC）裂解缓冲液（150mM的氯化铵 ，10毫KHCO 3，和1mM EDTA 35毫升，Pħ7.4）到含有新鲜血液样品（≥3的管:1体积/体积）并在室温（RT）下孵育不超过5分钟。 注:通过管透光的外观表示RBC裂解完成。 </li><li>离心在在RT 600 xg离心5分钟。确保粒料的颜色为白色。 </li><li>用10mL高压灭菌磷酸缓冲生理盐水的悬浮颗粒（PBS，137 mM氯化钠，2.7毫摩尔的KCl，4.3毫摩尔的 Na 2 HPO 4，和1.47毫摩尔KH 2 PO 4; pH7.4）和离心机如在步骤1.3。 </li><li>弃去上清液，用10ml的RPMI 1640培养基（补充10％胎牛血清，100U / mL青霉素/链霉素，0.25微克/毫升两性霉素B，和2mM L-谷氨酰胺）重悬沉淀，然后镀细胞分化成一个10厘米的培养皿（10毫升每皿血）。放置在37℃培养箱，5％CO 2的30分钟的盘。 </li><li>轻轻地摇晃培养皿几次，放置培养基（悬浮细胞）到15毫升的塑料锥形管。丢弃在培养皿中贴壁细胞（主要是巨噬细胞）。 </li><li>离心在在RT 600 xg离心5分钟。弃去上清液。 </li><li>悬浮用1mL的RPMI 1640培养基中的小球。计数与血球或自动细胞计数器的细胞数。 注:分离白细胞存活率通常是大于90％通过台盼蓝排除。 </li><li>离心机如在步骤1.7和5的浓度重悬在冷的PBS缓冲液（0.5％牛血清白蛋白（BSA）和2mM EDTA）中的〜200μL的细胞- 10×10 6个细胞/ mL。 </li><li>添加5μL的每10 6个细胞的特异性血细胞（B细胞的负选择）生物素化的抗-人抗体鸡尾酒的，并在冰上孵育30分钟21。 注:抗人抗体鸡尾酒至少应包括具体到CD2（或CD3），CD14，CD16和腹肌。 </li><li>添加10倍过量体积无菌PBS的细胞，离心分离，在600×g离心5分钟，并弃去上清液。 </li><li>添加链霉抗缀合的微珠等量（每10 6个细胞5微升）到颗粒并充分混合。 </li><li>在一个15毫升的塑料锥形管在冰上孵育30分钟。 </li><li>加入2毫升的PBS缓冲液到管。 </li><li>放置管进入一个磁性底座，并在室温下孵育8分钟。棕色微珠将逐渐附着在管壁的磁铁22旁边。 </li><li>与残留在磁性支架上的管中，小心地将上清液转移到一个新的无菌的15毫升管22。 </li><li>重复步骤1.14至1.16，并结合包含不变的B细胞两个上清液。丢弃微珠。 </li><li>离心在在RT 600 xg离心5分钟。弃去上清液。 </li><li>重悬在RPMI 1640培养基中的小球用于下游实验。 注意:通常情况下，1 - 5×10 5个 B细胞机智公顷纯度大于95％，从10毫升外周血可以分离23。 </li></ol> 2.分离的B细胞纯化和记忆的分离和普通B细胞<ol><li>使用从步骤1纯化的细胞，并确定使用血细胞计数器或自动细胞计数器的细胞数。 </li><li>重悬在100微升冷PBS缓冲液中的细胞在600 xg离心离心和RT 5分钟后。 </li><li>添加1 - 2每10 6个细胞的生物素化的CD27单抗的微克，并在冰上孵育30分钟。 </li><li>在600 XG添加10毫升的PBS缓冲液的管中，离心5分钟。 </li><li>弃上清，重悬细胞在PBS缓冲液50微升。 </li><li>添加链霉抗磁微珠的等量（1 - 2微克/ 10 6个细胞）的细胞在15毫升的塑料锥形管中。 </li><li>轻轻拌匀，在冰上孵育30分钟。 </li><li>添加2 - 3毫升PBS缓冲液到管。 </li><li>放置围成一个磁性底座和孵化在室温进行8分钟，以允许棕色微珠附着到最靠近磁体的一侧。 </li><li>与在磁性支架上的管中，小心地转移上清液部分到一个新的无菌试管中。该馏分包含富集CD27 -幼稚B细胞。 </li><li>添加5毫升含有微珠的管，轻轻悬浮微珠（ 即，CD27 +记忆B细胞的富集级分）24-25。 </li><li>离心在在RT 600 xg离心5分钟。重悬为下游实验的RPMI 1640介质中的颗粒。 注意:通常情况下，约30 - B细胞的60％，可以从健康供体7，25-26的PBMC中的CD27 +记忆细胞纯化。 </li></ol> 3.细胞为普通B细胞，记忆B细胞和PBS / PC的收集整理<ol><li>使用从步骤1纯化的细胞，确定使用hemocytome细胞数之三或自动细胞计数。 </li><li>重悬在冷的PBS缓冲液将细胞以10 7每毫升的5毫升聚苯乙烯管的浓度。 </li><li>添加1 - 2每10 6个细胞的人IgG微克，并在冰上孵育10分钟与Fc块。 </li><li>添加1微克的每一个抗CD19-APC（克隆:HIB19），抗CD27-eFluor450（克隆:O323），和抗CD38-PE（克隆:HIT2）每10 6个细胞;拌匀，在冰上孵育30分钟4，27。 </li><li>在步骤3.4的最后5分钟，加入商业7氨基放线菌素D（7-AAD）的5微升。 </li><li>在600 XG添加2毫升的PBS于管，涡旋，离心5分钟。 </li><li>悬浮细胞在1的浓度分拣缓冲液（无菌PBS用2％BSA和2mM EDTA） -在一个15毫升管每毫升5×10 7个细胞。 </li><li>过滤细胞通过尼龙网细胞滤网（40微米孔径），以消除细胞团块。 </li><li>分离细胞用流式细胞排序器配备了三个激光器:紫（405纳米），蓝色（488纳米），和红色（640纳米）。 注:单独的蓝色激光进行三色流式细胞27-28流量就足够了。 </li><li>排序细胞成三个15-mL管（含有5 RPMI培养基中的溶液），用于幼稚B细胞的同时采集（CD19 + CD27 - ），记忆B细胞（CD19 + CD27 +），和PBS /个人电脑（CD19 + CD27 + / HI CD38 +）3-4，28。 注:在步骤4中所述排序天真和记忆B细胞可以培养。 </li></ol> 4. 在离体人CD19 + B细胞，CD19 + CD27 +记忆B细胞和CD19 + CD27 体外分化-普通B细胞<ol><li>利用在步骤1.19，2.10，2.11和3.10纯化的细胞，确定与血球或自动细胞计数器的细胞数。 </li><li>悬浮用RPMI 1640培养基中的细胞以1:1的浓度- 10×10 5个每毫升和等分它们放入一个12孔板的孔中。 </li><li>在5微克/ 10 6细胞添加CPG（2006年ODN）/毫升18，29。 </li><li>培养的细胞在37℃培养箱，用5％ 的 CO 2 5天。 </li><li>收获来自每个细胞孔，分别将它们放入15-mL管中，加入5毫升的PBS至每个管，并在600 xg离心和RT 5分钟离心它们。 </li><li>算使用血球或自动细胞计数器细胞。悬浮细胞以1:1的浓度- 10×10 5个每毫升的RPMI 1640培养基。 </li></ol> 5.酶联免疫斑点检测<ol><li>在蒸馏水中添加35％乙醇的30微升到的ELISpot平板的每个孔中进行30秒。 注:吹打时，请避免在任何时候都接触在孔膜。 </li><li>倒置的ELISpot板以除去乙醇。 </li><li>把150蒸压DDH 2 O的的微升到每口井和孵化他们在RT 5分钟以冲洗掉残留的乙醇;按照与在RT无菌PBS洗涤3分钟。 注意:步骤5.1至5.3可以是可选的，这取决于板的制造中。 </li><li>放5微克/毫升的多克隆的F（ab&#39;）抗人Ig抗体（IgG + IgM阳性的IgA）2片段的50微升（在PBS中）插入的ELISpot平板的每个孔中，并在4℃下孵育他们过夜（优选的）或37℃2小时11。 注意:用Parafilm密封板的边缘，直到使用。 </li><li>倒置平板在RT以除去未结合ABS，添加的PBS 200微升至每个孔，并孵育他们两次，每次3分钟。 </li><li>添加的PBS200μL的含5％牛血清白蛋白（或RPMI1640培养基），以每孔用于阻挡，并在室温下孵育他们2小时。 </li><li>倒置平板以除去封闭缓冲液。洗各孔用PBS，200μL的两倍，在步骤5.5。 </li><li>加入100微升的RPMI 1640培养基中，以每个孔，并在37°C孵育它们。 </li><li>当准备种子细胞，反转板卸下RPMI 1640培养基。 </li><li>种子100微升（5×10 4），50微升（2.5×10 4），和25微升（1.25×10 4）的细胞（从步骤1.19，2.10，2.11，3.10，和4.6）转换成的一个孔中酶联免疫斑点板。用RPMI 1640培养基，使体积达到150微升/孔。 注:最小电镀细胞的一个或两个2倍连续稀释的建议。 </li><li>孵育在37℃培养箱板用5％CO 2的10 - 14小时18。避免孵育期间移动所述板。 </li><li>倒置平板以除去细胞和RPMI 1640培养基。 </li><li>加入200微升/孔的PBS-T（PBS含0.05％吐温20），并培育它们的5倍于RT，每次3分钟。反转板，除去每个5洗涤的洗涤液。 </li><li>添加任一的山羊抗人IgG-碱性磷酸酶（AP），与Fcγ特异性ABS（IgG的检测，1:5000于PBS-T的）或山羊抗人IgM-AP，Fcμ片段特异性ABS（对于IgM的检测，1:5000于PBS-T）到指定的孔，并在室温孵育在黑暗中2小时。 </li><li>洗各孔用PBS，200μL的两倍，在步骤5.5。 </li><li>添加50μL/孔的溴氯吲哚磷酸硝基蓝四唑（BCIP / NBT）底物溶液。紫色的斑点通常出现在5 - 15分钟。 </li><li>加入100微升/孔的DDH 2 O操作防止斑点的过度发展。 </li><li>与所有景点的全面发展后，自来水冲洗板。 </li><li>取出板的暗渠，并允许他们在黑暗中空气干燥。 </li><li>用自动板读数器与图像采集/分析单元（ 例如，一个自动扫描仪或通过在解剖显微镜手动）计数斑点。 注:该板可以存储在RT在黑暗中和后进行分析。 </li></ol>

The author declares no competing financial interests.

分离和人外周血B细胞的分离纯化通常情况下，红细胞可以有效地破裂并通过裂解缓冲液（步骤1.2）清零。它不孵育用RBC裂解缓冲超过5分钟长的PBMC是重要的，因为细胞生存力可能由氯化铵的影响。可替代地，RBC和血小板可以同时通过以下方案除去。 1，血液到缓冲液:在9:1的体积比;混合新鲜全血酸 - 柠檬酸盐 - 葡萄糖（ACD）缓冲液（pH 7.4 39 mM柠檬酸，75mM柠?...

B细胞发挥体液免疫的发展中发挥中心作用。他们最初开发在骨髓和进入血液流中作为幼稚B细胞，其可以迁移到淋巴组织，诸如脾，淋巴结，扁桃体，为进一步发展。在银相遇，一些幼稚B细胞迁移到淋巴滤泡，其中生发中心B细胞可以分化成记忆B细胞和plasmablasts（PBS）/浆细胞（PC）的。虽然大多数PBS / PC的出口进入血流，少数最终驻留在骨髓经受终端分化为长寿命的计算机1。 B细胞在循环是异构的，并且在稳定状态下，PBS / PC是外周血2罕见。作为谱系特异性表面标志物的可用性的结果，流式细胞术已经成为外周血B细胞亚群的鉴定和表征的常用方法。流动cytometr的扩展应用y是在加入细胞分选仪的功能，其允许分离与B细胞的个别亚群的分离具有高纯度的。基于人类循环B细胞一般分为三个主要亚群特异性表面受体在不同的发育阶段的表达:幼稚B细胞（CD19 + CD27 - CD38 - ），记忆B细胞（CD19 + CD27 + CD38 - ）和PBS /电脑（CD19 + CD27 + CD38 +）3-4（ 图1）。普通B细胞的性质还没有遇到过AGS。然而，它们可以分化成的IgM + CD27 +记忆B细胞。虽然幼稚B细胞在表达B细胞抗原受体（BCR）相关分子（ 例如，CD19，CD20和CD22），他们在他们的免疫球蛋白库5异构均匀。大多数CD27 +记忆B细胞可以分化成CD27+ / HI CD38 + PBS /电脑6。此外，记忆B细胞和PBS / PC是多克隆并表现出发育和功能的异质性4-7。流通中的PB / PC通常短暂的，不表达CD138，但那些由落户骨髓将最终分化，成为长寿。终末分化的PC表达CD138，下调其表面8 CD27分子。因为两者的PB和PC是能够分泌腹肌的，在许多情况下，他们被共同表示为携带者。相比之下，无论是普通B细胞，也没有记忆B细胞能产生相当数量的绝对9-10。放置在适当的培养条件下6，11-15时10日-然而，分离时，两者幼稚和记忆B细胞可以分化成的ASCs 3。事实上，从携带者和CD27 CD38 在体外分化有着相似的表面衍生的表达与那些直接分离FROM外周血6。此外， 在体外分化的携带者表达CD20表面的较低水平，这同样循环PBS /个人电脑6。虽然培养衍生的ASCs都是短暂的，它们能分泌腹肌，这表明它们在功能上是胜任和能够向体液免疫。 既ELISA和酶联免疫斑点是目前最常用的方法，用以获得关于体液免疫应答功能信息。 ELISA是一个96孔基于板的测定法，它经常用于测量血清抗原特异性ABS和其他分析物（ 例如，细胞因子）的效价。这是方便和可扩展性。 ELISA被设计为使用固相酶测定来检测ABS或其它物质，如血清存在下，液体样品16英寸从血清的ELISA所述读数已被广泛地用于表示身体的免疫反应。必要收购重的工具从ELISA检测adouts是酶标仪。读者可以决定最终产品通常由辣根过氧化物酶（HRP）缀合的检测ABS和其特定的底物17的反应所得的光密度（OD）。对于报告的体液免疫应答，通过ELISA测定血清抗体水平表示在身体的ASC的集体，但不是个别，性能。此外，酶联免疫吸附未能顾及由记忆B细胞，其不分泌腹肌的参与。 像酶联免疫吸附，酶联免疫斑点是用于检测和监测外周血样品17-18中免疫应答的广泛使用的方法。酶联免疫斑点是涉及夹心ELISA的技术。在它，将细胞放置到聚偏二氟乙烯（PVDF）的96孔膜支持孔微孔板用于短期培养。 ELISPOT试验类似于上微孔板和developin进行免疫印迹克，以每孔中的PVDF膜的斑点。一种自动化的ELISpot读取器系统或人工计数立体显微镜是必需的。酶联免疫斑点的检测的免疫应答的主要优点是在携带者和细胞因子分泌细胞的定量其极好的灵敏度。它分别报告它们的功能活动，体液免疫和细胞免疫。在体液免疫功能的测定中，通过ELISA和通过ELISPOT列举的ASC的数量来确定血清抗体水平通常相关，但来自这两个测定法中的数据读出有功能的影响19-20一些差异。酶联免疫斑点的主要优点是它的方法的灵敏度。血清抗体滴度通过ELISA报告的水平呈现半定量作为OD读数，表示相对抗体水平，或更定量地，作为集中读数当包括参考腹肌的适当的同种型的已知量。与此相反，酶联免疫斑点的结果是presente-d作为携带者的在感兴趣的细胞池的绝对数量（ 例如，未分级的外周血单核细胞（PBMC），并从PBMC中纯化的B细胞）。酶联免疫斑点法可以检测单个ASC，但ELISA需要来自的ASCs抗体达之前测量达到优化的测定依赖浓度。因此，酶联免疫斑点是定量的灵敏度明显优于ELISA。此外，酶联免疫斑点法也适用于量化从激活记忆B细胞的体外分化的携带者。记忆B细胞不分泌腹肌但能分化成在激活时的ASC;于是他们就用ELISA法检测血清中绝对没有任何贡献。因此，酶联免疫斑点是选择在培养活化后循环记忆B细胞的免疫应答的测定方法。它允许对维持长期体液免疫的监测。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD Vacutainer K2E</td>
			<td>BD Biosciences</td>
			<td>367525</td>
			<td>10 ml tube</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td>endotoxin-free</td>
		</tr>
		<tr>
			<td>Trypan blue 0.5% solution</td>
			<td>Biological Industries</td>
			<td>03-102-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>IMag Human B lymphocyte enrichment set</td>
			<td>BD Biosciences</td>
			<td>558007</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated CD27 mAb</td>
			<td>Biolegend</td>
			<td>302804</td>
			<td>clone O323</td>
		</tr>
		<tr>
			<td>Streptavidin magnetic microbeads</td>
			<td>BD Biosciences</td>
			<td>9000810</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Falcon tubes</td>
			<td>BD Falcon</td>
			<td>352196</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue nylon mesh cell strainer, 40 &#956;m</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD19-APC</td>
			<td>Biolegend</td>
			<td>302212</td>
			<td>clone HIB19</td>
		</tr>
		<tr>
			<td>Anti-human CD27-eFluor 450</td>
			<td>eBioscience</td>
			<td>48-0279-42</td>
			<td>clone O323</td>
		</tr>
		<tr>
			<td>Anti-human CD38-PE-Cy7</td>
			<td>Biolegend</td>
			<td>303516</td>
			<td>clone HIT2&#160;</td>
		</tr>
		<tr>
			<td>Anti-human CD38-PE-Cy7</td>
			<td>BD Biosciences</td>
			<td>560677</td>
			<td>clone HIT2&#160;</td>
		</tr>
		<tr>
			<td>Anti-human CD45-FITC</td>
			<td>Biolegend</td>
			<td>304006</td>
			<td>clone HI30</td>
		</tr>
		<tr>
			<td>Anti-human CD45-FITC</td>
			<td>BD Biosciences</td>
			<td>555482</td>
			<td>clone HI30</td>
		</tr>
		<tr>
			<td>Anti-mouse/rat/human CD27-PerCP Cy5.5</td>
			<td>Biolegend</td>
			<td>124213</td>
			<td>clone LG.3A10</td>
		</tr>
		<tr>
			<td>Anti-human CD27-PerCP Cy5.5</td>
			<td>BD Biosciences</td>
			<td>65429</td>
			<td>clone L128</td>
		</tr>
		<tr>
			<td>Anti-human CD19-FITC</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-064</td>
			<td>clone LT19</td>
		</tr>
		<tr>
			<td>Anti-human CD19-FITC</td>
			<td>GeneTex</td>
			<td>GTX75599</td>
			<td>clone LT19</td>
		</tr>
		<tr>
			<td>Anti-human CD20-FITC</td>
			<td>BD Biosciences</td>
			<td>555622</td>
			<td>clone 2H7</td>
		</tr>
		<tr>
			<td>biotinylated anti-human CD27</td>
			<td>Biolegend</td>
			<td>302804</td>
			<td>clone O323</td>
		</tr>
		<tr>
			<td>biotinylated anti-human CD27</td>
			<td>eBioscience</td>
			<td>13-0279-80</td>
			<td>clone O323</td>
		</tr>
		<tr>
			<td>7-aminoactinomycin D (7-AAD)</td>
			<td>BD Biosciences</td>
			<td>559925</td>
			<td></td>
		</tr>
		<tr>
			<td>CpG (ODN 2006)&#160;</td>
			<td>InvivoGen</td>
			<td>tlrl-2006</td>
			<td>type B CpG</td>
		</tr>
		<tr>
			<td>Recombinant human IL-2</td>
			<td>PeproTech</td>
			<td>200-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-10</td>
			<td>PeproTech</td>
			<td>200-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-21</td>
			<td>PeproTech</td>
			<td>200-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human sCD40L</td>
			<td>PeproTech</td>
			<td>310-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein A of S. aureus Cowan (SAC)</td>
			<td>Sigma-Aldrich</td>
			<td>82526</td>
			<td></td>
		</tr>
		<tr>
			<td>Pokeweed mitogen (PWM)</td>
			<td>Sigma-Aldrich</td>
			<td>L9379</td>
			<td></td>
		</tr>
		<tr>
			<td>MultiScreen filter plates, 0.45 &#181;m pore size</td>
			<td>Merck Millipore</td>
			<td>MSIPS4510</td>
			<td>sterile, clear 96-well filter plate with hydrophobic PVDF membrane</td>
		</tr>
		<tr>
			<td>BCIP/NBT solution</td>
			<td>Sigma-Aldrich</td>
			<td>B6404</td>
			<td></td>
		</tr>
		<tr>
			<td>BCIP/NBT single reagent, alkaline phosphatase substrate</td>
			<td>Merck Millipore</td>
			<td>ES006</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>009-000-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IgG, Fc fragment</td>
			<td>Jackson ImmunoResearch</td>
			<td>009-000-008</td>
			<td></td>
		</tr>
		<tr>
			<td>F(ab&#39;)2 fragment of goat anti-human Ig (IgG+IgM+IgA)</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-006-127</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-human IgG-alkaline phosphatase, Fc&#947; fragment specific</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-055-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-human IgM-alkaline phosphatase, Fc&#181; fragment specific</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-055-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-human IgG-peroxidase, Fc&#947; fragment specific</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-035-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-human IgM-peroxidase, Fc&#181; fragment specific</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-035-095</td>
			<td></td>
		</tr>
		<tr>
			<td>BD ELISPOT AEC substrate kit</td>
			<td>BD Biosciences</td>
			<td>551951</td>
			<td></td>
		</tr>
		<tr>
			<td>C.T.L. ImmunoSpot analyzer</td>
			<td>C.T.L.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the isolation, differentiation, and quantification of human antibody-secreting b cells from blood: elispot as a functional readout of humoral immunity

体液免疫的特点是，以产生功能性的ASC，其合成和分泌特异性抗原（银），ABS等的病原体，并用于宿主防御。用于定量测定的个体的体液免疫应答的功能状态，既血清ABS和循环的ASC通常为功能读数测定。在人类中，外周血是可用于由主机B细胞引起的体液免疫应答的确定最方便和最容易获得的样本。不同B细胞亚群，包括的ASC，可直接从外周血通过选择与谱系特异性抗体共轭微珠或通过细胞仪具有流量分选分离。此外，纯化的幼稚和记忆B细胞可被激活并分化成在培养的ASCs。的ASCs促进抗体分泌的功能活性可以通过ELISPOT进行量化，这是收敛酶的测定法- 连接immunoabsorbance吸附试验（ELISA）和Western印迹技术，使个人的ASC的枚举在单细胞水平。在实践中，ELISPOT测定已越来越多地用于评估，因为易于大量血液样品的处理的疫苗效力。从外周血中分离的人类B细胞的方法中，B细胞的分化为携带者，和酶联免疫斑点的就业总IgM-和IgG-的ASC的定量将在这里描述。

外周血单个核细胞被耗尽的红细胞和贴壁细胞（步骤1.2到1.7）的。细胞的等分试样（2×10 6）进行流式细胞分析来说明幼稚B细胞，记忆B细胞和外周血（ 图1）PBS / PC的人口。在这个供体的外周血单个核细胞，淋巴细胞的约10％是CD19 + B细胞。在B细胞室，CD19 + CD27的百分比-幼稚B细胞是在50％左右。另一方面，所述CD19 + B细?...

Watch this Scientific Journal Video about The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity at JoVE

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

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 ...

the-isolation-differentiation-quantification-human-antibody-secreting

Research

JoVE Journal

Immunology and Infection

15.1K 次观看.  National Taiwan University. 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.

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

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

小鼠腹膜腔细胞的分离

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

科研

免疫与感染

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

紧凑型超支化聚乙二醇接枝的膜抗原受保护的功能性红血细胞

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

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 ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

对白细胞从流程的分析招聘基质细胞的影响

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

分离，鉴定和牙龈免疫细胞网络的功能性考试

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34+ cells. A method for CD34+ cell isolation and megakaryocyte differentiation is described here.

人脐血巨分化与血小板形成的 CD34+单元格

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...