我们感谢华盛顿大学医学院和巴恩斯犹太医院在圣路易斯，密苏里州的阿尔文J. Siteman癌症中心，为使用高速细胞分选的核心，提供流式细胞仪检测设备和排序服务。这项工作是由国家资助卫生研究院R01 HL082549和K02 HL084179（NRY）的支持。 Siteman癌症中心是支持部分由NCI癌症中心支援津贴P30 CA91842。

1。试剂的制备<ol><li>准备无菌FMS相关酪氨酸激酶3的1000倍的股票解决方案（FLT - 3）配体，粒细胞/巨噬细胞集落刺激因子（GM - CSF的），干细胞因子（SCF），血小板生成素（TPO），白细胞介素（IL）-3 ，IL - 6和根据制造商的指示。准备的无菌Retronectin股票溶液（1mg/ml的）也可根据制造商的说明。分成小等份，这些股票的解决方案，并保持在-20 ° C，避免反复冻融。 </li><li>准备2％FBS的IMDM。 </li><li>准备完整的IMDM培养液终浓度20％胎牛血清，谷氨酰胺2毫米，100个单位/ ml青霉素/链霉素（PS）。使用前加入以下因子：100毫微克/毫升FLT - 3配体，20 ng / ml的GM - CSF 100 ng / ml的SCF的，100 ng / ml的TPO，50 ng / ml的IL - 3，和100纳克/毫升IL - 6。 </li><li>准备1％BSA的HBSS（钙和镁的自由，无酚红）和2％的牛血清白蛋白在D - PBS（钙和镁免费）。通过0.22微米针头式过滤器和存储在4 ° C。的消毒解决方案</li><li>准备在0.02％EDTA的HBSS，并储存在4 ° C。 </li><li>准备镀锌假型逆转录病毒编码的DNA分装的兴趣和存储于-80 ° C。此过程是本文的范围之外，但其他地方描述（1-3）。 </li></ol>对于姬姆萨染色<ol start="7"><li>使用前，通过1微米到一个干净的玻璃瓶的滤纸过滤两次赖特/姬姆萨染色解决方案。 </li><li>使用前，准备赖特/吉姆萨缓冲液pH 6.4，混合50毫升的过滤赖特/姬姆萨染色溶液和200毫升磷酸盐缓冲液，pH值6.4 - 佐丹奴公式。 </li><li>准备通过溶解在3.5 LH 2 O KH 2 PO 4 27.3克和4.62克的Na 2 HPO 4的磷酸盐缓冲溶液，pH值6.0 </li><li>准备在H 2 O 50％甲醇</li></ol> 2。程序解冻和预激活的CD34 +细胞<ol><li>一小瓶冷冻CD34 +细胞迅速解冻，在37 ° C时轻轻晃动​​，直到最后的小冰晶左侧和转移的细胞悬液（1毫升）50毫升锥形管。轻轻冲洗掉1毫升室温从瓶中剩余的细胞2％FBS / IMDM培养，并把它添加落明智的，50毫升管轻轻纷飞。等待3分钟。接下来，慢慢加入2毫升2％FBS / IMDM，同时轻轻搅拌，并等待3分钟。重复添加2％FBS / IMDM是相同体积稀释的细胞悬液，在3分钟的间隔，轻轻纷飞之间增加，直到最终的量达到32毫升的过程。 </li><li>在室温为10分钟，在250 XG离心，去除上清液，留下约0.5毫升。 </li><li>洗净颗粒悬浮在20毫升2％FBS / IMDM培养和离心8分钟，在室温200 XG。 </li><li>去除上清液，并暂停完整的IMDM培养基中的细胞 ，约0.5 × 10 6细胞/ ml。考虑到一个微管10μL的细胞悬液，混合相同体积的台盼蓝溶液（0.4％）和使用一个血球计数。 </li><li>加入与细胞因子为辅的完整的IMDM培养基稀释细胞0.1-0.15 × 10 6细胞/ ml（ 见 1.3） </li><li>文化与5％的CO 2天2细胞中的饱和湿度37℃培养箱。 </li></ol>预加载病毒的逆转录病毒转导<ol start="7"><li>转天，堆载预压，以确定井数要准备开始前病毒的细胞进行计数。 </li><li> 25微克/毫升Retronectin的原液稀释，添加400μL，每孔24孔非治疗组织培养板。层流生物安全罩前大衣为2小时，在室温下孵育。 </li><li>取出Retronectin的解决方案，并添加400μL的2％BSA的D - PBS，每孔。 30分钟，在室温下孵育。 </li><li>解冻病毒的解决方案，并保持在冰上。删除BSA溶液，每孔加入0.5毫升病毒编写，并在4℃离心2200 XG 15分钟。 </li><li>从水井中删除病毒的解决方案和重复病毒负荷在2.10三次描述。 </li><li>每孔0.5毫升冷的HBSS（+钙和镁）或IMDM冲洗。 </li><li>在室温下8分钟200 XG离心收集预激活的CD34 +细胞。 </li><li>重悬在0.1〜0.15 × 10 6个细胞与细胞因子的完整的IMDM培养液1.5毫升的细胞。 </li><li>加入1.5 ml的细胞悬液，每孔和5％的CO 2天2饱和湿度37℃培养箱中孵育。 </li></ol>细胞分类和收集细胞<ol start="16"><li>轻轻地，但彻底中止细胞病毒在上面装板S和过滤器通过50微米CellTrics成一个锥形管的细胞过滤器。台盼蓝溶液等体积混合成微管，以10μL的细胞悬液，计数使用一个血球。冲洗每口井用0.8毫升冷0.02％EDTA / HBSS（钙和镁无酚红），并通过50微米CellTrics细胞过滤器添加到同一个收集管。 </li><li> 8分钟，在4 ° C离心200 XG的细胞，颗粒（钙和镁无酚红）与冷的HBSS洗离心8分钟，在4 200 XG ° C。 </li><li>在1％BSA / HBSS（钙和镁无酚红）15 × 10 6 / ml或体积最小的细胞分类为0.250毫升的浓度重悬沉淀。继续在黑暗中的冰的细胞。准备一个试管中冷20％FBS / IMDM /谷氨酰胺/ PS收集排序细胞的培养基。排序是细胞的阿尔文J. Siteman癌症中心在华盛顿大学医学院的医学使用一个MoFlo高速分拣机（DAKO，Glostrup，丹麦）高速分拣机的核心。 </li></ol>氟氯化碳含量测定<ol start="19"><li>解冻所需数量的氟氯化碳检测媒体3毫升分装。涡大力混合，并让管站至少5分钟，让目前任何气泡上升之前加入细胞表面。 </li><li>要获得每个排序的样品细胞浓度，划分分类设施，提供近似的细胞悬液体积的细胞数。以3000病毒转排序，进入无菌冷2％FBS / IMDM中含微管的细胞。预调整量的2％FBS / IMDM培养等，最终悬浮液体积约0.3毫升。 </li><li>悬浮细胞和整个0.3毫升细胞悬液转移到3毫升分装Methocult GF + H4435，其中包括1％甲基纤维素，30％FBS，1％BSA，10 -4 M 2 -巯基乙醇，2毫米L -谷氨酰胺， 50毫微克/毫升的SCF，20 ng / ml的GM - CSF的，20 ng / ml的IL - 3，20 ng / ml的IL - 6，20毫微克/毫升G - CSF和3个单位/毫升红细胞生成素的IMDM。人力甲基纤维素丰富的媒体（R＆D系统）1.3％甲基纤维素组成，25％FBS，1％BSA，5 × 10 -5 M 2 -巯基乙醇，2毫米L -谷氨酰胺，50纳克/毫升的SCF，20毫微克/毫升GM - CSF的，20 ng / ml的IL - 3，20 ng / ml的IL - 6，20毫微克/毫升G - CSF和3个单位/毫升的IMDM促红细胞生成素可也使用。 </li><li>涡大力充分混合上升和下降3-4倍。让管站3分钟。 </li><li> 3毫升注射器将16计钝针，并制定了2.2毫升。不要画了大泡;驱逐他们开始推了几次。推了1.1毫升，每一个到两个30毫米非治疗的菜和蔓延旋转混合均匀。 </li><li>放置在一个100毫米的钢板重复板块包含3毫升无菌水的水菜。文化为14 - 17天。 </li><li>根据其标记具有得分电网培养皿放大40倍倒置显微镜的形态特点和得分殖民地。对于我们实验的目的，菌落分为3类：纯红，骨髓和混合。请参见制造商的指示，为进一步殖民地subclassification。 </li><li>可能没有使用一个定期在600 dpi的扫描仪，低功耗（40X）菌落显微照片可采取配备彩色摄像机使用倒置显微镜的放大倍率扫描整个氟氯化碳检测板。 </li><li>整个氟氯化碳检测板的细胞分化和增殖的进一步分析，恢复暂停在室温2％FBS / IMDM中的几卷。在300 XG在4 ° C 10分钟离心后，细胞悬浮在2％FBS / IMDM计算，或者染色，流式细胞仪检测抗体，或转移到幻灯片使用一个Cytospin离心机姬姆萨染色。 </li></ol>抗体染色和流式细胞仪<ol start="28"><li> 8时4分在200 XG离心机细胞° C和重悬在寒冷的50％正常小鼠血清中的HBSS（钙和镁无酚红）。 </li><li>放置在每管80μL（12 × 75毫米圆底管）约5 × 10 5细胞，并保持在冰上。添加抗体混合物（约20μL混合，或无抗体控制阴性对照溶液）和混合轻轻拍打管。准备与无抗体或抗CD45抗体，每个荧光校准控制管。在黑暗中孵育20分钟冰放置管。混合管后第10分钟，轻轻拍打。 20分钟潜伏期后，填写与冷的HBSS管和离心8分钟在200 XG，在4 ° C。在寒冷的0.5％的多聚甲醛/的HBSS重悬沉淀。 我们的分析通常用于以下抗体的混合物。髓样分化：细胞CD11b（藻红蛋白共轭克隆ICRF44）/ CD33（allophycocyanin共轭克隆WM53）/ CD45（藻红蛋白- Cy7 congjugated克隆HI30）。红细胞分化：CD71（藻红蛋白共轭克隆的M - A712）/ CD235a（变藻蓝蛋白偶联克隆的GA - R2）/ CD45（藻红蛋白- Cy7 congjugated克隆HI30）。 </li><li>的阿尔文J. Siteman癌症中心在华盛顿大学医学院的细胞的高速分拣机核心升级到5种颜色和两个激光器（BD Biosciences公司）在FACScan流式细胞流式细胞仪和流式细胞仪进行分析使用CellQuest（BD Biosciences公司）或FlowJO v7.2.4（树星公司，亚什兰，美国）软件。 </li></ol>姬姆萨染色<ol start="31"><li>以上所述的从氟氯化碳的检测板回收约30,000细胞暂停在0.3毫升2％FBS / IMDM培养和转移到幻灯片使用一个Cytospin离心机在室温下10分钟1000转。 </li><li>设置了九个染色血管，含有下列顺序解决方案。 <ol><li>绝对甲醇</li><li>赖特/姬姆萨原液</li><li>赖特/吉姆萨缓冲液，pH值6.4 </li><li>在H 2 O的 50％甲醇</li><li> H 2 O </li><li> H 2 O </li><li>磷酸盐缓冲液，pH值6.0 </li><li> H 2 O </li><li> H 2 O </li></ol></li><li>放入幻灯片和幻灯片的载体，浸在绝对甲醇（＃1），2分钟吸干多余的甲醇。 </li><li>随即，浸5分钟，赖特/姬姆萨原液幻灯片载波（＃2）。 </li><li>转移到赖特/吉姆萨缓冲液，pH 6.4（＃3）承运人和孵育10分钟。 </li><li>浸在50％甲醇的载体两次（＃4），10倍，在H 2 O（＃5），另外的10倍，H 2 O（＃6），然后在磷酸盐缓冲液，pH值6.0的5倍（＃ 7） 。 </li><li>转移到H 2 O（＃8）承运人和孵育2分钟。在H 2 O（＃9）重复洗。 </li><li>允许的幻灯片空气干燥的载体，完全。取出每张幻灯片，幻灯片用甲醇浸泡Kimwipes背面擦拭去除污渍。将下降了60 Cytoseal和一个密封的玻璃盖。 </li><li>一个500细胞分类计数是每个姬姆萨染色使用奥林巴斯BX51显微镜幻灯片。细胞分为五大类：原始细胞，包括爆炸和promyelocytes;中间的骨髓细胞，包括myelocytes和metamyelocytes;成熟的骨髓细胞，包括乐队，分段中性粒细胞，单核细胞和巨噬细胞，中间的红系细胞，包括中间hemoglobinization的细胞和成熟的红系细胞细胞与完整hemoglobinization。显微照片是采取了奥林巴斯DP71相机60X石油目标。 </li></ol> 3.0）代表性的成果： <ol><li> 氟氯化碳含量测定：输入细胞群的增殖，分化，和半固体培养基上形成菌落，在14 17天的潜伏期。在这个实验中，这是显而易见的，NUP98 - HOXA9癌基因的表达导致形成突出的红色（红殖民地）（图1）（3）。 </li><li> 抗体染色和流式细胞仪流式细胞免疫分型：流量从CFC检测板收集的细胞分化状态的细胞群提供的信息。对某些分化标记的抗体结合使用，宗族和细胞的成熟程度进行评估。在这种情况下，被用来确定CD235a红系细胞。如图2a所示，NUP98 - HOXA9造成CD235a +红系细胞的比例增加，但这些细胞的亮度CD235a表达减少，与对照组相比，说明抑制红细胞的成熟。在图2b中，凭借其CD33阳性骨髓细胞识别。 NUP98 - HOXA9导致在CD33 +细胞的数量减少，在CD11b表达的亮度下降，抑制髓性成熟（3）一致。因此，流式细胞仪检测结果显示，NUP98 - HOXA9造成抑制髓细胞和红细胞的成熟的红细胞增生。 </li><li> Giemsa染色：从CFC检测板收集的细胞形态学检查，细胞群的成熟程度和宗族的信息。流式细胞仪的研究中获得的形态获得的结论可能会有所不同，因此这些方法都是相辅相成，相互（3）。实验至少重复3次，每次500细胞分类计数进行区分5个类别，细胞如上所述。这些细胞的例子指出，在图3。结果列和分析，以确定是否有显着性差异癌基因转导的样品和控制。在这种情况下，结果表明，NUP98 - HOXA9造成一个整体的数量增加的细胞，红细胞增生及抑制红和髓系成熟（3）（未显示）。 </li></ol>图例<img src="/files/ftp_upload/2195/2195fig1.jpg" alt="图1" /> 图1：对人类氟氯化碳形态NUP98 - HOXA9的影响 。 首要人权CD34 +细胞retrovirally转要么控制MSCV - IRES - GFP的向量或向量表示NUP98 - HOXA9，和排序的GFP阳性细胞。一千个细胞接种到每个两个重复板氟氯化碳检测，实验重复3 4独立的时间。代表板无放大（左）和代表红殖民地（右）的低功耗显微照片（3）所示。 <img src="/files/ftp_upload/2195/2195fig2.jpg" alt="图2" /> 图2：流式细胞仪检测显示NUP98 - HOXA9人类首要的CD34 +细胞分化受阻。 （一）流式细胞仪检测红细胞分化：从CFC板的细胞（见图1）收获与抗体染色，CD45和CD235a。 （二）的CD235a +门上绘制直方图（右图），显示相对于对照组细胞表达CD235a流式细胞仪检测髓样分化：从CFC板收获细胞和CD45和CD33染色。 CD33 +门上绘制直方图来显示CD11b表达与对照组比较（右图）。每个门范围内的细胞的百分比（3）所示。 <img src="/files/ftp_upload/2195/2195fig3.jpg" alt="图3" /> 图3：细胞形态显示NUP98 - HOXA9分化的破坏相比，矢量控制 。 从CFC板Cytospin涂片，Giemsa染色。显微照片取自代表一个60倍的石油的目标领域。 B：爆炸，MM：成熟的粒细胞，即时通讯：中间髓系，ME：成熟的红细胞，即：中间红（3）。

<ol>
	<li>Takeda, A., Goolsby, C., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NUP98-HOXA9+induces+long-term+proliferation+and+blocks+differentiation+of+primary+human+CD34++hematopoietic+cells.">NUP98-HOXA9 induces long-term proliferation and blocks differentiation of primary human CD34+ hematopoietic cells.</a> Cancer Res. , 66-6628 (2006).</li><li>Yassin, E. R., Abdul-Nabi, A. M., Takeda, A., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+NUP98-DDX10+oncogene+on+primary+human+CD34++cells:+role+of+a+conserved+helicase+motif.">Effects of the NUP98-DDX10 oncogene on primary human CD34+ cells: role of a conserved helicase motif.</a> Leukemia. , (2010).</li><li>Yassin, E. R., Sarma, N. J., Abdul-Nabi, A. M., Dombrowski, J., Han, Y., Takeda, A., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+the+transformation+of+primary+human+hematopoietic+cells+by+the+oncogene+NUP98-HOXA9.">Dissection of the transformation of primary human hematopoietic cells by the oncogene NUP98-HOXA9.</a> PLoS One. 4, e6719-e6719 (2009).</li><li>Nissen-Druey, C., Tichelli, A., Meyer-Monard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hematopoietic+colonies+in+health+and+disease.">Human hematopoietic colonies in health and disease.</a> Acta Haematol. 113, 5-96 (2005).</li><li>Pereira, C., Clarke, E., Damen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+colony-forming+cell+assays.">Hematopoietic colony-forming cell assays.</a> Methods Mol Biol. 407, 177-208 (2007).</li><li>Coulombel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+hematopoietic+stem/progenitor+cells:+strength+and+drawbacks+of+functional+assays.">Identification of hematopoietic stem/progenitor cells: strength and drawbacks of functional assays.</a> Oncogene. 23, 7210-7222 (2004).</li><li>Hogge, D. E., Lansdorp, P. M., Reid, D., Gerhard, B., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+detection,+maintenance,+and+differentiation+of+primitive+human+hematopoietic+cells+in+cultures+containing+murine+fibroblasts+engineered+to+produce+human+steel+factor,+interleukin-3,+and+granulocyte+colony-stimulating+factor.">Enhanced detection, maintenance, and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human steel factor, interleukin-3, and granulocyte colony-stimulating factor.</a> Blood. 88, 3765-3773 (1996).</li></ol>

已广泛用于氟氯化碳检测，以确定造血祖细胞的增殖和分化的模式，并研究原癌基因的影响（4，5）。它利用液体培养的克隆实验，殖民地代表一个单系祖的后代，并作进一步的分析可以单独删除，。的CFC检测的限制，这是不足够更不成熟的祖细胞或造血干细胞的检测，发现这种细胞的长期培养启动细胞（LTC - IC）的测定（6，7）。这里所描述的实验表明，反洗钱相关基因NUP98 - HOXA9抑制髓系和红系...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>IMDM</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>12440</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>06150</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin (PS)</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>FLT-3 ligand</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-19</td>
<td>&nbsp;</td>
<tr>
<td>GM-CSF</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-03</td>
<td>&nbsp;</td>
<tr>
<td>SCF</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-07</td>
<td>&nbsp;</td>
<tr>
<td>TPO</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-18</td>
<td>&nbsp;</td>
<tr>
<td>IL3</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>200-03</td>
<td>&nbsp;</td>
<tr>
<td>IL6</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>200-06</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma- Aldrich</td>
<td>A7030</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP118</td>
<td>&nbsp;</td>
<tr>
<td>Retronectin</td>
<td>&nbsp;</td>
<td>Takara Bio Inc</td>
<td>T100A</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>14175</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>14200</td>
<td>&nbsp;</td>
<tr>
<td>Trypan Blue solution (0.4%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T8154</td>
<td>&nbsp;</td>
<tr>
<td>Methocult GF+ H4435</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>04445</td>
<td>&nbsp;</td>
<tr>
<td>Human Methylcellulose Enriched Media</td>
<td>&nbsp;</td>
<td>R &amp; D Systems</td>
<td>HSC005</td>
<td>&nbsp;</td>
<tr>
<td>Wright/ Giemsa stain</td>
<td>&nbsp;</td>
<td>Harleco</td>
<td>64571</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffer Solution, pH 6.4 - Giordano formula</td>
<td>&nbsp;</td>
<td>Ricca Chemical Company</td>
<td>1450</td>
<td>&nbsp;</td>
<tr>
<td>Methanol</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>A412-4</td>
<td>&nbsp;</td>
<tr>
<td>Cytoseal 60</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>8310</td>
<td>&nbsp;</td>
<tr>
<td>Normal Mouse Serum </td>
<td>&nbsp;</td>
<td>Rockland Immunochemicals</td>
<td>D208</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD11b phycoerythrin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>555388</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD33 allophycocyanin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>551378</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD45 phycoerythrin-Cy7-congjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>557748</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD71 phycoerythrin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>555537</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD235a allophycocyanin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>551336</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD45 phycoerythrin-Cy7-congjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>557748</td>
<td>&nbsp;</td>
<tr>
<td>24-well non-treated tissue culture plates</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-1147</td>
<td>&nbsp;</td>
<tr>
<td>30 mm non-treated dish</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>27150 </td>
<td>&nbsp;</td>
<tr>
<td>100 mm tissue culture dish </td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>Gridded scoring dishes</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>27500</td>
<td>&nbsp;</td>
<tr>
<td>15 ml centrifuge tubes</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-2097</td>
<td>&nbsp;</td>
<tr>
<td>50 ml centrifuge tubes</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-2070</td>
<td>&nbsp;</td>
<tr>
<td>Syringes 3 ml </td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>28240</td>
<td>&nbsp;</td>
<tr>
<td>16 gauge blunt-end, 1&frac12; inch needle </td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>28110</td>
<td>&nbsp;</td>
<tr>
<td>50 &mu;m CellTrics cell filter</td>
<td>&nbsp;</td>
<td>Partec</td>
<td>04-004-2327</td>
<td>&nbsp;</td>
<tr>
<td>Hemocytometer</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>0267110</td>
<td>&nbsp;</td>
<tr>
<td>TPX sample chambers</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>A78710018</td>
<td>&nbsp;</td>
<tr>
<td>Fisherbrand Superfrost/Plus Microscope Slides, Precleaned</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-550-15</td>
<td>&nbsp;</td>
<tr>
<td>Shandon filter cards</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>5991022</td>
<td>&nbsp;</td>
<tr>
<td>Shandon cytospin slide holder</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>59920063</td>
<td>&nbsp;</td>
<tr>
<td>Shandon Complete Staining Assembly 100</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>100</td>
<td>&nbsp;</td>
<tr>
<td>Kimwipes</td>
<td>&nbsp;</td>
<td>Kimberly-Clark Corporation</td>
<td>34155</td>
<td>&nbsp;</td>
<tr>
<td>1 &mu;m filter paper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>28307-134</td>
<td>&nbsp;</td>
<tr>
<td>Inverted microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>Diaphot</td>
<td>&nbsp;</td>
<tr>
<td>Microscope camera</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>DS-F11</td>
<td>&nbsp;</td>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>BX51 </td>
<td>&nbsp;</td>
<tr>
<td>Microscope camera</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>DP71</td>
<td>&nbsp;</td>
<tr>
<td>Scanner</td>
<td>&nbsp;</td>
<td>Microtek</td>
<td>Scanmaker 4</td>
<td>&nbsp;</td>
<tr>
<td>Vortex mixer</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-812</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture incubator</td>
<td>&nbsp;</td>
<td>Sanyo</td>
<td>MCO-18AIC</td>
<td>&nbsp;</td>
<tr>
<td>Cytospin</td>
<td>&nbsp;</td>
<td>Shandon</td>
<td>Cytospin 2</td>
<td>&nbsp;</td>
<tr>
<td>Bench-top centrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5810-R</td>
<td>&nbsp;</td>
<tr>
<td>Water purification system</td>
<td>&nbsp;</td>
<td>Barnstead</td>
<td>Nanopure-Diamond</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

colony forming cell (cfc) assay for human hematopoietic cells

人造血干/祖细胞通常从骨髓，脐带血，外周血和用于研究造血和白血病。他们有能力分化成淋巴和髓细胞谱系。集落形成细胞（CFC）的测定是用来研究的增殖和分化的造血祖细胞的能力，在半固体培养基中形成的殖民地模式。一个固定数量的输入细胞形成的菌落的数量和形态提供初步信息有关的祖细胞能够分化和增殖。从单个菌落或从整个板块收获细胞可以进一步评估它们的数量和分化状态，用流式细胞仪检测和形态学评估姬姆萨染色幻灯片。这种分析是有用的评估髓细胞分化，但没有淋巴。在这方面的长期髓系在其更广泛的意义上使用，包括粒细胞，单核细胞，红系，巨核细胞谱系。 我们已经用这种检测评估的首要人权从外周血CD34 +细胞衍生的细胞分化癌基因的影响。这样做的目的细胞转要么控制逆转录病毒构建或利益表达的原癌基因的构建，在这种情况下，NUP98 - HOXA9。我们采用一种常用的逆转录病毒载体MSCV - IRES - GFP，表达了产生的利益和GFP标记基因的双顺反子mRNA的。细胞被预先激活，越来越多的前两天，以逆转录病毒转导的细胞因子的存在。另外两天后，GFP +细胞是孤立的荧光激活细胞分选（FACS），并含有一个甲基纤维素半固体与细胞因子的补充和培养至菌落出现在表面上，一般为14天的中期混合。记录的数量和形态的殖民地。细胞，然后从板块中删除，洗涤，计数，和流式细胞仪检测和形态学检查。流式细胞仪检测表达在造血细胞表面标记的特异性抗体提供有关血统和成熟阶段的信息。 Wright - Giemsa染色后显微镜下单个细胞的形态学研究提供进一步的信息方面的血统和成熟。控制空载体转导细胞比较，揭示造血分化癌基因的影响癌基因转导。

Watch this Scientific Journal Video about Colony Forming Cell CFC Assay for Human Hematopoietic Cells at JoVE.com

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

集落形成细胞（CFC）的检测是在体外实验中，造血祖细胞形成半固体培养基中的菌落。结合菌落形态，细胞形态，流式细胞仪是用来评估的祖细胞的增殖和分化沿不同的造血谱系能力。

集落形成细胞（CFC）的含量对人体的造血细胞

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

colony-forming-cell-cfc-assay-for-human-hematopoietic-cells

Research

JoVE Journal

Immunology and Infection

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

35.5K 次观看.  Washington University School of Medicine. 集落形成细胞（CFC）的检测是在体外实验中，造血祖细胞形成半固体培养基中的菌落。结合菌落形态，细胞形态，流式细胞仪是用来评估的祖细胞的增殖和分化沿不同的造血谱系能力。

视频: Colony Forming Cell CFC Assay for Human Hematopoietic Cells

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

科研

免疫与感染

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&amp;#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

使用γ-干扰素酶酶联免疫检测的特点人类乳头状瘤病毒的新型T-细胞表位

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

在纯化造血干/祖细胞识别DNA突变

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

杀手人工抗原提呈细胞（KaAPC）为有效<em&gt;在体外</em&gt;人抗原特异性T细胞耗竭

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

巨噬细胞集落刺激因子分化的人单核细胞衍生的巨噬细胞的培养

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

人外周血 NK 细胞活性评估流基于流式细胞仪检测细胞毒性检测

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...