这项工作是由美国国立卫生研究院（NCI R00 CA132784）KT

1。脐血单个核细胞分离<ol><li>准备EDTA的PBS缓冲液中添加5毫升牛血清白蛋白（BSA）的储备溶液至95毫升漂洗缓冲液（1:20稀释）。德加的缓冲和保持缓冲区冰。 重要提示：未脱气缓冲隔离时，可能会导致在不到最佳的效果CD19 + B细胞，因为气泡会阻止隔离柱。 </li><li>准备50毫升锥形管密度梯度离心法。确定所需的处理的脐带血管的数目（1管可以处理血8毫升），并在每个管中加入15毫升的Ficoll-Paque联合PLUS。 </li><li>用24毫升的DPBS（1X），并仔细层稀释脐血混合物在每个50 ml锥形管的顶部，用Ficoll-Paque联合PLUS稀释8毫升脐带血。不要混合血液和帕克的Ficoll-PLUS。 重要提示：为了避免混合脐带血和帕克的Ficoll-PLUS，保持在45度角的管层的血液混合物缓慢。 </li><li>在20℃下离心40分钟，在400×g离心单核细胞（MNC）将保持在等离子体的Ficoll-Paque联合PLUS接口，而粒细胞和红细胞的沉积物由于更高的密度的Ficoll-Paque PLUS的渗透压。出版商7 5 ml圆底使用的钢管，在与该样本ID，日期，和下面的第3部分： </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> 管 </td><td> 标签 </td><td> 目的 </td></tr><tr><td> 1 </td><td>未染色的</td><td>未染色流式细胞仪检测细胞正常化</td></tr><tr><td> 2 </td><td> 7AAD </td><td>要确定在流式细胞仪的可行性</td></tr><tr><td> 3 </td><td> + + + + </td><td>包含将被排序的细胞</td></tr><tr><td> 4 </td><td> 34 + </td><td>为了收集CD34 + / CD19 +（晚B - 提前预BI）细胞</td></tr><tr><td> 5 </td><td> 45 低 </td><td>为了收集CD34 - / CD19 + / CD45 低 （BI）细胞</td></tr><tr><td> 6 </td><td> 45 MED </td><td>为了收集CD34 - / CD19 + / CD45 MED细胞（前BII） </td></tr><tr><td> 7 </td><td> 45 高 </td><td>为了收集CD34 - / CD19 + / CD45 高 （未成熟的B细胞） </td></tr></tbody></table>外套管4，5，6，和7与2％FBS和所有的管子放置在冰上。 <ol start="5"><li>吸上的等离子层，仔细，避免与单核细胞层接触。使用10毫升玻璃吸管小心转移到一个新的50毫升锥形管中的单核细胞层。的单核细胞从三个管结合成一个单一的50毫升的试管一起。 </li><li>填充管，PBS，轻轻混匀，在300 XG离心10分钟，20°C。小心吸出上清液，不干扰细胞沉淀。重复1倍。在第一次洗涤后，悬浮颗粒，并转移到一个单一的50毫升锥形管。 </li><li>轻轻重悬细胞沉淀，在200μl的PBS中。删除1微升的细胞悬液，加入1毫升的PBS在1.5 ml离心管中，并预留计数（计数细胞后，将50 ml细胞悬浮液在离心机中）。用PBS填充管，并在200×g离心15分钟以除去血小板离心。完全去除上清，不干扰细胞沉淀。 </li></ol> 2。改进的B-细胞使用MACS分离的单个核细胞的分离<ol><li>从1.7步为160μl冷（4℃），EDTA-PBS缓冲液重悬沉淀。 </li><li>加入40μlB-CLL生物素的抗体鸡尾酒。移液管拌匀，于4℃孵育10分钟</li><li>洗涤细胞 - 加1毫升冷（4℃），EDTA-PBS缓冲液，每10万个单元格（单元格NU人數确定步骤1.7）中，并在300×g离心10分钟，离心。完全吸取上清，然后在320微升冷（4℃）EDTA的PBS缓冲液中重悬细胞沉淀。 </li><li>加入80微升抗生物素微球，搅拌均匀，于4℃孵育15分钟</li><li>洗涤细胞 - 添加1ml冷的（4℃）EDTA的PBS缓冲液，每10万个细胞，在300×g离心10分钟离心。在低温（4℃）EDTA-PBS缓冲液500μL重悬到100万个细胞。根据细胞数的比例缓冲液体积。 </li><li>准备MACS柱和MACS分离器在离心过程中（步骤2.5）。一个LS柱放置在磁场的MACS分离器中，加入3毫升冷的（4℃）EDTA的PBS缓冲液中，在色谱柱上，并允许在缓冲区中的通过柱滴落。使用一个插座，以赶上流过。丢弃流过。 </li><li>栏下方放置一个50毫升锥形管和移液到柱上的细胞悬浮液。允许未标记的细胞滴通过省h的色谱柱并进入50毫升锥形管。重要信息：这些将用于抗体标记的细胞分选的细胞。不要丢弃流过。为了恢复所有的细胞从步骤2.5，添加额外的1毫升的冷（4℃）EDTA的PBS缓冲液中的锥形管的壁。轻轻混匀列上移液剩余的细胞悬浮液。重复1倍。收集在锥形管中使用的标记的细胞通过柱后，继续到步骤2.8。 </li><li>填充的锥形管，含有未标记细胞在300×g离心10分钟（用PBS）和离心机。小心地取出，而不会干扰细胞沉淀，取上清液。加入200μl的EDTA-PBS缓冲液中，并轻轻悬浮细胞。 </li></ol> 3。抗体标记细胞分选和准备<ol><li>管标记为“未染色的”（步骤1.4）的细胞悬浮液，并放入吸液1微升。加入500μlEDTA的PBS缓冲液，并在冰上储存管。 </li><li>加入20微升每种抗体（CD19，CD34和CD45）每百万个细胞的到其它细胞悬浮液。充分混匀，并放置在黑暗中30分钟，在RT管。 CD抗体是光敏感的，完整的步骤2-4关灯。 </li><li>抗体孵育30分钟后，吸取40微升细胞悬浮液，并将其放置到管标记为“7AAD”。到管中并离心，在500×g离心2分钟，加入1毫升的PBS。除去上清液，加入100μl的结合缓冲液中重悬细胞。加入7微升7AAD（7 - 氨基放线菌素D），并在黑暗中孵育10分钟，在RT。在孵育10分钟完成步骤3.4。 </li><li>加入PBS中含有剩余的细胞标记的抗体与CD的管的顶部。离心管中于500×g离心3分钟。除去上清液，加入500μlEDTA的PBS缓冲液中重悬细胞沉淀。的整个体积的细胞悬浮液转移到管中，标记为“+ + +”。要恢复所有的细胞悬浮液中西元一个额外1毫升EDTA-PBS缓冲液的50毫升锥形管和管转移到标有“+ + +”。 </li><li>孵化后（步骤3.3），加入300μl结合缓冲液到7AAD管。 “不染”，“7AAD”和“+ + +”样本和“34 +”，“45低 ”，“45 MED”和“45 高 ”管准备流式细胞仪检测。 </li></ol> 4。使用的MoFlo XDP流式细胞仪细胞分选<ol><li> SET-UP MoFlo排序：对准激光器，稳定液滴流，确定截止延时。 </li><li>准备根据制造商的说明使用Invitrogen ABC鼠标磁珠试剂盒（或类似）的单一色彩补偿控制。运行单一颜色控制，调整荧光通道的电压，以获得最佳分离阳性和阴性的人群。设置补偿系数，适用于收集协议的补偿参数。重新建立补偿系数每一次得到一个新的大量的抗体。 </li><li>创建协议包括如在图2中示出的图。 </li><li>运行未染色的样品，正向和侧向散射光的设置电压和增益，并确定负荧光人口。由于DNA恢复目标的排序，应设置阈值低（≤1％），因此，DNA的碎片不会污染回收的样本。 *确保在任何时候，生活被上运行的MoFlo的人体细胞的气溶胶疏散系统正在运行。 </li><li>运行一个小的分量+ + +样品（约50,000个事件），设置的门控策略（ 图2）。由于B-细胞的祖细胞不分散等同于成熟的B细胞，背栅非门CD19 + / CD34 +人口，到SSC和FSC情节的，以确保淋巴细胞门，包括潜在的原B细胞。从这个示例还设置负的荧光人口为7AAD。 </li><li>运行的7AAD的的样本，以确定样品的可行性。只有细胞的样品具有高生存能力的排序一开始（≥95％），死亡的细胞会弄脏不分青红皂白地和可能污染排序人群。 </li><li>设置排序的决定，收集四个群体：CD19 + / CD34 +，CD19 + / CD34 - / CD45 低 ; CD19 + / CD34 - / CD45 MED和CD19 + / CD34 - / CD45 很高的 。包括在排序的决定的所有人群的的淋巴细胞和双峰歧视门。 </li><li>在分拣小费为防止堵塞，过滤器+ + +示例通过40μm紧接排序和重新过滤，排序，如果没有聚合发生在样本期间之前的细胞过滤网。 </li><li>分类的细胞到涂覆有2％FBS的在PBS中，在冷却的或冰的包装管夹持器的收集管（步骤1.4）。 </li><li>排序后立即完成后，提取DNA。 </li></ol>

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作者没有透露。

用的协议的成功的影响最大的因素是存在污染的碎片。如果请求的帘线血库的血液从它重要的是，有血液采集后尽快装运尽可能。此外，样品被归类为淋巴细胞增多含有较高数量的淋巴细胞;然而，这些样品不具有足够数目的前体B细胞，不应该使用。为了增加获得足够数量的细胞，我们建议至少85毫升脐带血开始的每一个的前体的子集的概率。 重要的是要注意，不像成人外周血分离时，分馏脐带血单核层往往是与红血细胞12污染。该协议包括一个额外的清洗步骤，以去除污染的血小板。协议描述了从脐带血单核分离建议，包括裂解步骤TØ删除不需要的红细胞。我们不建议这一步，因为它产生污染的碎片和的单元格进行排序的成功有负面影响。 为了减少数量的细胞，通过流式细胞术来加以区分，它是必要的执行之前的B-细胞富集细胞分拣。的协议，伴随着B-细胞分离试剂盒（B-CLL），由美天旎生物技术公司，提供优化的外周血，并建议使用10μL的B-CLL生物素的抗体鸡尾酒每10万个细胞。此步骤使用抗CD2（T细胞，NK细胞），CD4（T细胞），细胞CD11b（粒细胞，单核细胞，巨噬细胞），CD16（NK细胞，巨噬细胞，肥大细胞），CD36（血小板），CD235a（红系细胞）耗尽非B细胞从脐带血。重要的是要使用所述的试剂盒，而不是B细胞分离试剂盒II，因为前者套件包含CD43的抗体，它是存在于原B细胞。在我们的运timization这个协议，我们发现，共有40微升的B-CLL生物素的抗体鸡尾酒足以产生阳性细胞的排序结果。此外，我们发现，使用B-CLL的生物素抗体鸡尾酒低至5微升的单元格进行排序产生了不利的影响。因此，我们强烈建议您使用40μL的B-CLL生物素的抗体鸡尾酒单核细胞数在175-250万美元之间。如果开始的较低或较高的细胞数，可能有必要相应缩放试剂。 有许多相互矛盾的出版物描述的标记，可用于区分B-细胞的亚群的。大多数的差异可以通过以下说明的事实，即分化是一个持续的过程中，细胞表面标志物的存在或不存在下在渐进的方式，而不是在一个全有或全无的方式发生。在这个协议中的CD34 - / CD19 +和CD45 +的强度水平（低，中，高）的表达被用来区分预BI，预-BII和未成熟的B细胞的CD45的表达水平对应于分化6的进展增加。前B细胞已被有些人所描述的是CD19 + / CD34 + 13，而另一些则表明，前B细胞CD19 - / CD34 +前BI细胞CD19 + / CD34 + 9,10。基于这些差异，我们已指定为已故亲B细胞/的早期预BI细胞CD19 + / CD34 +细胞。重要的是要注意，这种策略的开发是为了隔离前体B细胞，对应于受疾病的急性淋巴细胞白血病细胞的子集。但是，也有多个分类策略取决于感兴趣的细胞亚型，也可以采用。 抗体 - 荧光染料的组合的选择应基于可用的细胞分选仪的能力。作为一个属升规则，应使用面板中最亮的荧光标记人口最少的抗原，反之亦然。在双染人群，尤其是罕见的事件，如检测，双重歧视（ 图2B）是一个非常重要的部分，门控策略。这可确保双阳性的事件是真正的单一的细胞与抗体和不只是两个单染色的细胞附着到另一个染色。 高速，4路细胞分选必然创建环境受控气溶胶。因此，人类活细胞分选应非常谨慎。发布的安全和净化的指引，应审查和执行排序前14。批准之前可能需要从生物安全委员会或其等值排序。排序后的适当的去污，漂白剂，应添加至终浓度为10％到废液容器。硅milarly，所有的样品管以及在邻近地区的所有表面应彻底清洁，新鲜的10％的漂白粉溶液。 总之，这个协议提供了一个获得难得的前体B细胞的人口和战略进行修改，以隔离罕见的人口存在于脐带血造血干细胞和未成熟的T细胞。最近，未成熟的B细胞已被确定外周血中的个人与先进的艾滋病毒15。因此，这种方法的实用程序延伸超出血液癌症的研究。最后，我们还尚未执行的流上的排序条件的细胞的RNA的隔离，但是，这种方法应该是适应的警告，在细胞分选的细胞应直接进入三唑或等效RNA兼容的解决方案，如RLT排序从RNeasy试剂​​盒可通过Qiagen公司。

为了识别异常疾病的存在，这是非常重要的，我们用健康的组织或细胞受该疾病影响的组织或细胞类型相对应的。其中一个原因是，负责调控基因表达的表观遗传变异之间的组织类型是至关重要的细胞分化过程中正常的人类发展1,2。第二个原因是，异常的组织特异性基因的调控可能会造成严重的后果正常发展，并有助于包括癌症在内的多种疾病状态。因此，更好地了解造血干细胞的疾病，它涉及需要健康的造血干细胞的知识。 造血干细胞在骨髓中通过系统其特征在于，在细胞表面表达的变化的事件顺序所得的发展标记3。研究涉及成人学员如图，骨髓通常包含一个低的前体B细胞4,5;而涉及儿童参加的研究表明，B-细胞前体的比例是比较高的，在不到5岁6个人。血液相关的疾病和恶性肿瘤的治疗中被用作脐带血的造血干细胞的来源，是现成通过脐带血的银行，并且富集未成熟的B细胞和T细胞的7，它的靶细胞的多种疾病，包括白血病和淋巴瘤。 前体B细胞在骨髓中已被广泛的QTL 8,9和由成不同的子集，可以用于分类，这些细胞的特定的细胞表面标志物的存在下，可以定义。正常的B细胞分化开始与最早的原B细胞在骨髓中和最终在不成熟或幼稚的B细胞通过一系列的阶段所得。据车ZELM和他的同事10其特征在于，原B细胞的CD34的存在，和在第2阶段的过渡到（预BI）CD19获取。第3阶段（前BII）细胞不再表达CD34和，开始表达胞质IgM抗体。最后，第4阶段（未成熟的B细胞）的定义性特征是表面IgM的表达。在该协议中描述的排序策略首先描述由Caldwell和他的同事6和包括只有3细胞表面标志物，从而大大降低了复杂性和成本进行细胞分选实验使用。在他们的工作中，CD45和B细胞分化的阶段之间的关系成立。他们观察到的B细胞在骨髓中显示可变的CD45的表达水平的。具体而言，细胞表达高水平的CD45细胞表达表面IgM（未成熟的B-细胞）相对应，这些表达的CD45的中间电平相对应的细胞表达cytoplasmic IgM的（预BII细胞），和那些表达低水平的CD45对应的细胞不表达细胞质的IgM（预BI细胞）。该协议使用的策略开发人员，Caldwell和他的同事们6隔离子集的前体B细胞从脐带血中（ 图1），可以用在需要高品质的核酸，如甲基化的CpG岛恢复测定的下游检测（MIRA ）11和定量实时PCR测定。的方法，采用使用磁珠耗尽所有非B细胞从脐带血中的初步分离，并要求只有3个抗体（CD34，CD19和CD45）染色。所回收的细胞代表B细胞分化的4个阶段：1）的CD34 + CD19 +（晚亲B和早期预BI）的2）的CD34 - CD19 +，CD45低 （晚期预BI）; 3）CD34 - ，CD19 +，CD45 MED（预-BII）和4）的CD34 - ，CD19 + CD45 高 （未成熟的B-细胞）。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DPBS (1X)</td>
			<td>Gibco by Life Technologies</td>
			<td>14190-144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Healthcare Bio-Sciences AB</td>
			<td>17-1440-03</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LS Column</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MACS Multi Stand</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MidiMACS Separator</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B-Cell Isolation Kit (B CLL)</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-093-660</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ATLANTA Biologicals</td>
			<td>S11195</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube (1.5 ml)</td>
			<td>MIDSCI</td>
			<td>SS1500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BD Pharmingen 7-AAD (7-Aminoactinomycin D)</td>
			<td>BD Biosciences</td>
			<td>559763</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DB Pharmingen APC Mouse Anti-Human CD19</td>
			<td>BD Biosciences</td>
			<td>555415</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DB Pharmingen PE Mouse Anti-Human CD34</td>
			<td>BD Biosciences</td>
			<td>560941</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CD45 FITC</td>
			<td>BD Biosciences</td>
			<td>347463</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>autoMACS Rinsing Solution</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MACS BSA Stock Solution</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BD Falcon 5 ml Polystyrene Round-Bottom Tube</td>
			<td>BD Biosciences</td>
			<td>352058</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BD Falcon 50 ml Tube</td>
			<td>BD Biosciences</td>
			<td>352098</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PuraFlow Sheath Fluid, 8X</td>
			<td>Beckman Coulter</td>
			<td>CY30230</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FlowCheck Alignment beads</td>
			<td>Beckman Coulter</td>
			<td>6605359</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ultra Rainbow Alignment beads</td>
			<td>Spherotech</td>
			<td>URFP-30-2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ViroSafe Aerosol Evacuation Filter</td>
			<td>Beckman Coulter</td>
			<td>ML01330</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ABC Mouse bead kit</td>
			<td>Invitrogen</td>
			<td>A-10344</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>40 &mu;m cell strainer</td>
			<td>Fisher Scientific</td>
			<td>22363547</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fisher Scientific Hemocytometer</td>
			<td>Fisher Scientific</td>
			<td>267110</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>accuSpin Model 3R Benchtop Centrifuge</td>
			<td>Fisher Scientific</td>
			<td>13-100-516</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MoFlo XDP flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td>ML99030</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aerosol Evacuation Unit</td>
			<td>Beckman Coulter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

isolation of precursor b-cell subsets from umbilical cord blood

脐带血造血祖细胞是高度浓缩在不同血统的承诺阶段。我们已经开发出了一个协议，用于在四个不同的分化阶段的B细胞前体隔离。由于基因的表达和表观遗传修饰发生在以组织特异性的方式，这是极为重要的，以便能够识别的基因组和表观基因的改变，可能会导致疾病的发展的组织​​和细胞类型来区分。此方法可适合于任何类型的细胞中存在的在任何阶段的分化脐带血。 该方法包括4个主要步骤。首先，单核细胞，通过密度梯度离心分离。二使用生物素共轭的抗体，该抗体识别和除去非从单核细胞中的B-细胞，B-细胞富集。第三次的B-细胞，荧光标记与特定于各个阶段的细胞表面蛋白的抗体B细胞的发育。最后，该荧光标记的细胞进行排序和个别人群中回收。将回收的细胞是下游核酸检测中要使用的足够的数量和质量。

样品之间变异的细胞排序（ 表1）的成功起到了重要作用。具有良好的成功率的样品污染的碎片有低的水平（ 图3A）和成功率差的样品具有高的水平的污染的碎片（ 图3B）。样品之间的变化可以在一定程度上控制，如果脐带血样本收集在24小时内装运（O / N优先）。 排序的流动从每个前体B-细胞亚群细胞是足够的数量和质量来执行核酸隔离。分离的DNA是高品质的，并可以用在下游分析。我们经常在MIRA 11利用该DNA甲基化后的DNA（ 图4），以丰富。 <table border="1" fo:keep-together.within-page="always"><tbody><tr><td> 造血干细胞储存设施数据 </td><td> 差排序 </td><td><st荣好人</td></tr><tr><td>工作血容量抗凝血剂（毫升） </td><td> 110 </td><td> 104 </td></tr><tr><td>白血细胞[10 3个 /μl] </td><td> 8.68 </td><td> 11.19 </td></tr><tr><td>淋巴细胞[10 3个 /μl] </td><td> 3.88 </td><td> 3.76 </td></tr><tr><td>淋巴细胞（％） </td><td> 44.70 </td><td> 33.6 </td></tr><tr><td>红血细胞[10 6个/μl] </td><td> 3.65 </td><td> 3.05 </td></tr><tr><td colspan="3"> 内部数据 </td></tr><tr><td>后细胞数的Ficoll-Paque </td><td> 206 M </td><td> 309米</td></tr><tr><td> MACS分离后的细胞数</td><td> 22 M </td><td> 16.5米</td></tr><tr><td> “事件总数的计数（MoFlo XDP） </td><td> 30.57中号</td><td> 19.97中号</td></tr><tr><td>存活率（％） </td><td> 98.00 </td><td> 98.00 </td></tr><tr><td>细胞的淋巴细胞门（％） </TD&gt; <td> 17.00 </td><td> 50.00 </td></tr><tr><td colspan="3"> CD19 + / CD34 + </td></tr><tr><td>总细胞数</td><td> 10959 </td><td> 48316 </td></tr><tr><td>事件总数的百分比</td><td> 0.04 </td><td> 0.24 </td></tr><tr><td>效率</td><td> 88％ </td><td> 88％ </td></tr><tr><td colspan="3"> CD19 + / CD34 - / CD45 低 </td></tr><tr><td>总细胞数</td><td> 16619 </td><td> 26941 </td></tr><tr><td>事件总数的百分比</td><td> 0.05 </td><td> 0.13 </td></tr><tr><td>效率</td><td> 89％ </td><td> 87％ </td></tr><tr><td colspan="3"> CD19 + / CD34 - / CD45 MED </td></tr><tr><td>总细胞数</td><td> 469745 </td><td> 507540 </td></tr><tr><td>事件总数的百分比</td><td> 1.54 </td><td> 2.54 </td></tr><tr><td>效率</td><td> 89％ </td><td> 89％ </td></tr><tr><td colspan="3"> CD19 + / CD34 - / CD45 很高的 </td></tr><tr><td>总细胞数</td><td> 1896062 </td><td> 2047142 </td></tr><tr><td>事件总数的百分比</td><td> 6.2 </td><td> 10.25 </td></tr><tr><td>效率</td><td> 91％ </td><td> 90％ </td></tr></tbody></table> 表1中。代表性的细胞排列的统计数据。行1-5提供脐带血设施的计数。其余行是在我们的实验室中收集的数据。可怜的排序含有高含量的碎片和细胞的淋巴细胞门的比例很低。 <img alt="图1" src="/files/ftp_upload/50402/50402fig1.jpg" /> FiguRE 1。处理程序的流程图。脐带血单核细胞分离使用的Ficoll-Paque联合加。包括一个额外的洗涤步骤，以除去污染的血小板。 B-细胞，分离出单核细胞，使用的MACS人B-细胞分离试剂盒（B-CLL）。此步骤中使用生物素标记的单克隆抗体（CD2，CD4，CD36，CD11b和抗-IgE和CD235a），以除去所有非B细胞。回收的B-细胞标记CD19-APC，CD34-PE，CD45-FITC抗体，然后排序使用的MoFlo XDP流式细胞仪检测，恢复前体B细胞亚群：CD19 + / CD34 +，CD19 + / CD34 - / CD45 低 CD19 + / CD34 - / CD45 MED，和CD19 + / CD34 - / CD45 很高的 。 <img alt="图2" src="/files/ftp_upload/50402/50402fig2.jpg" /> 图2。门控策略，识别和排序弹出ulations红色箭头表示的栅极被施加到随后的情节。 R4，R5，R6和R7的门显示的排序条件种群。a）远期-侧散点图示出富集淋巴细胞群。 R1，淋巴细胞门。b）高度与宽度的前散点图确定单个细胞与双峰。 R2，单细胞的大门。 三）CD19-APC阳性细胞分为CD34-PE阴性和阳性的人群。 R3，CD19 + / CD34 -门; R4，CD19 + / CD34 +门表示所需的排序人口D）CD19 + / CD34 -细胞分为三种稍微不同的CD45-FITC人口的。 R5和R6，CD19 + / CD34 - / CD45 低 ，CD19 + / CD34 - / CD45，分别MED人群。 R7，因为细胞中的CD19 + / CD34 - / CD45 高人口数量，这种门仅包括的人口的一部分。所有细胞这一人口不进行下游分析需要收集。 <img alt="图3" src="/files/ftp_upload/50402/50402fig3.jpg" /> 图3：A）水平低，污染柱富集后的碎片。 R1，淋巴细胞门。b）高柱富集后的碎屑污染水平。 <img alt="图4" src="/files/ftp_upload/50402/50402fig4.jpg" /> 图4。富集的前体B-细胞的子集。）经声波处理的前体B细胞亚群的分离的DNA是被甲基化了的DNA的高品质。对于图书馆建设中的DNA进行超声处理的平均200-600个基点。第1道：Promega公司的100 bp的阶梯（目录＃G2101）;泳道2：分离出CD19 + / CD34 +细胞的总DNA，泳道3：总DNA分离出CD19 + </s升/ CD34 - / CD45 低细胞;四巷：100 ng的DNA分离出CD19 + / CD34 - / CD45 MED细胞;弄5号100 ng的DNA分离出CD19 + / CD34 - / CD45细胞。 DNA的超声处理使用Diagenode Bioruptor高温共9分钟（30秒，30秒OFF）。 DNA是柱纯化，并可视化，在1％琼脂糖凝胶电泳，用SYBR绿色核酸凝胶染色。 二）MIRA使用ActivMotif MethylCollector超试剂盒，PCR与SLC25A37（1-6）和APC1（7-12）后，进行确认富集的甲基化DNA。 1 - 7：声波处理的基因组DNA（没有MIRA），2＆8：MIRA-CD19 + / CD34 + DNA; 3和第9：MIRA-CD19 + / CD34 - / CD45 低 DNA; 4和10：MIRA-CD19 + / CD34 - / CD45 MED DNA; 5 - 11：MIRA CD19 + / CD34 - / CD45 DNA 高 ; 6和12：水的控制。高等扩增SLC25A37确认被甲基化了的DNA的富集的子集的前体B细胞。

Watch this Scientific Journal Video about Isolation of Precursor B-cell Subsets from Umbilical Cord Blood at JoVE.com

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

在这里，我们描述的协议子集的前体B细胞从脐带血中隔离开来。阿足够的数量和质量，可从细胞中提取核酸，并且用于在随后的实验，利用DNA或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-of-precursor-b-cell-subsets-from-umbilical-cord-blood

Research

JoVE Journal

Immunology and Infection

17.8K 次观看.  University of Missouri-Columbia. 在这里，我们描述的协议子集的前体B细胞从脐带血中隔离开来。阿足够的数量和质量，可从细胞中提取核酸，并且用于在随后的实验，利用DNA或RNA。

视频: Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

补消耗的特定细胞群的枯竭

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

科研

免疫与感染

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 ...

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

大脑和脊髓的单核细胞的分离，使用Percoll梯度

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

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 ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

临床应用<em&gt;睡美人</em和人工抗原呈递细胞基因修饰T细胞从外设和脐带血中

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Development of an IFN-&amp;#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

的IFN-γ酶联免疫斑点检测，以评估水痘 - 带状疱疹病毒特异性细胞免疫继脐血移植的发展

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

小鼠树突状细胞亚群的分离的高效和高产的方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

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 ...