作者承认澳大利亚卫生和医学研究理事会 (与六六六相关的项目赠款 1012409) 的支持。

该议定书得到了东南悉尼人类研究伦理委员会的批准, 并得到新南威尔士大学人类研究伦理委员会的批准。从健康捐献者获得的脐带血由悉尼脐血库 (悉尼, 新南威尔士州) 提供。这个过程使用了大约100毫升的容量。
注: 使用无菌技术的第二类生物安全柜工作。用70% 乙醇净化脐带血袋的外部。使用无菌器械 (剪刀、镊子) 进行此程序。
1. 脐血细胞的制备和分离 CD34 的+单元格
<ol>
	<li>用磷酸盐缓冲盐水 (PBS) 制备无菌分离缓冲液 (SB), pH 值为 7.2, 含0.5% 牛血清白蛋白和2毫米 EDTA。</li>
	<li>将10毫升的 SB 放入50毫升的锥形管中 (每10毫升的血液需要一根管子)。使用一个 G18 钝针安装在10毫升注射器, 绘制10毫升的血液从袋子和分配到50毫升管包含10毫升的 SB。</li>
	<li>添加15毫升的淋巴细胞分离介质 (参见材料表) 到含有稀释血液的管子底部, 形成两层。 注意: 在管子的底部慢慢地分配介质, 以避免混合不同的层。</li>
	<li>离心管在 1200 x g 为30分钟在室温 (RT) 没有断裂和没有加速度。</li>
	<li>将含有单个核细胞 (大约 5-10 毫升) 的管的中心附近的层转移到一个新的50毫升管中, 使用巴斯德吸管。每管增加一个, 总容积为50毫升。</li>
	<li>离心机在 400 x g 为10分钟, 在 RT. 丢弃上清。</li>
	<li>5-10 毫升的巴斯德重细胞颗粒小心地将悬浮细胞结合起来, 将体积带到50毫升。</li>
	<li>使用台盼蓝染色和例或自动单元计数器计数单元格 (请参见材料表)。要确定样本中的 CD34+单元格的百分比, 请在100µL 中重 2.5 x 105单元格, 并在10° c 中添加15µL anti-CD34-PE 抗体为 30-4 分钟。用流式细胞仪分析 (图 1A)。</li>
	<li>离心管在 400 x g 为 10 min 在4° c。丢弃上清。 注: 如果不需要立即, 单核细胞可以冻结在这个阶段。</li>
	<li>每 108单元格中的重单元格为300µL。添加100µL FcR 人 IgG (阻断试剂, 见材料表) 和100µL CD34 磁珠每 108细胞。轻轻混合, 孵育在4° c 30-40 分钟。 注: 需要单细胞悬浮 (如有必要, 在添加试剂前通过30µm 过滤器)。使用此处描述的试剂卷 108单元格或更少。对于超过 108的单元格, 应相应地向上扩展试剂 (SB、阻塞溶液和 CD34 磁珠)。</li>
	<li>将 ls 柱放在磁性支架上, 用3毫升的水冲洗, 在潜伏期内准备 ls 分离柱。 注意: LS 分隔列可以加载最多 2 x 108总单元格。更小的和更大的专栏是可利用的。</li>
	<li>将 SB 的 5-10 毫升加入到细胞混合物中, 在 400 x g 上离心10分钟, 丢弃上清液。</li>
	<li>在1.5 毫升的 SB 中重细胞, 并将细胞悬浮 (1.5 毫升) 加载到 LS 柱上。收集在15毫升收集管 (负分数 #1) 中含有未标记细胞的流动。让液体排出, 用1.5 毫升的水冲洗柱子。</li>
	<li>将收集到的流量通过 (3 毫升) 重新加载到该列中, 并在相同的15毫升收集管 (负分数 #1) 中收集流经。用3毫升的 SB 清洗 LS 列3次。 注意: 如果需要, 负分数 (12 毫升) 中的细胞可以用 anti-CD34 抗体染色, 以确定 LS 列捕获 CD34 的+细胞。</li>
	<li>从磁性分离器中取出柱, 用注射器柱塞慢慢冲洗细胞, 用2毫升的新的15毫升管。</li>
	<li>将列放回磁选机上, 并将2毫升的单元格重新加载到列中。收集2毫升的水流和洗涤。这是负分数 #2 (4 毫升)。</li>
	<li>从磁选机中取出 LS 柱, 然后用注射器柱塞稳定地和固定地加2毫升冲洗细胞, 以收集 CD34 的+单元格分数。使用台盼蓝染色和例或自动电池计数器计数细胞。</li>
	<li>离心管与正部分在 400 x g 为 15 min 在4° c。丢弃上清液。重细胞在无血清的介质中用于 CD34+单元格 (可持续性, 请参阅材料表)。 注: 如果不需要立即, 细胞可以冻结在液氮在这个阶段。</li>
</ol>
2. 隔离 CD34+单元格的纯度检查
<ol>
	<li>染色 2 x 104细胞从阳性分数与10µL anti-CD34-PE 抗体和20µL anti-CD45-PerCP 抗体15分钟在4° c (使用一个单独的管同种控制) (图 1B)。</li>
	<li>加1毫升的 SB 洗。离心机在 400 x g 为10分钟的重颗粒在200µL 的 SB。</li>
	<li>执行流细胞分析, 并将活体细胞填充到排除碎片 (图 1B)。使用 pe 和 PerCP 同种控件 (图 1B) 设置 pe 和 PerCP 阳性填充的门, 并确定 CD34+/CD45+单元格的百分比。</li>
</ol>
3. 巨分化
<ol>
	<li>种子 5 x 105细胞/ml CD34+细胞在2毫升的可持续的可持续的可持续的可持续利用 50 ng/毫升重组人生成 (rhTPO) 每井在 12-井板。在湿润的气氛中孵育细胞在37° c, 5% CO2 。如果细胞是汇合的, 然后才需要分析, 收获的细胞和分裂成多个井与新鲜的媒体和 rhTPO。 注: 应专门准备两个或三口井, 以监测不同时间点的差异 (例如、天7、9和 10)。</li>
	<li>从油井中抽出的细胞来监测分化, 而不干扰其他水井中的细胞。</li>
	<li>染色细胞与20µL anti-GPIIb/CD41-FITC 抗体和10µL anti-GPIX/CD42a-Alexa 氟647抗体在最后的体积100µL. 使用各自的同种控制抗体设置控制管。孵育 15-30 分钟在4° c。</li>
	<li>加1毫升的 SB 洗。</li></ol>离心机在 400 x g 10 分钟, 丢弃上清, 重 200-300 µL 中的颗粒. 流式细胞仪分析。
	<li>对于每个荧光, 分析同种控制, 设置 FITC 和 Alexa 面粉647阳性人群的浇口。分析染色单元样本以确定 CD41+/CD42a+双阳性单元格的百分比, 它们代表成熟的 MK (图 1C)。</li>
	<li>用于细胞表面标记染色细胞的显微可视化, 如3.3 所述。<ol>
		<li>加1毫升的 SB 洗。离心机在 400 x g 为10分钟. 重100µL 中的球团, 旋转到 1000 x g 的玻片上, 用甲醇浸泡在三十年代的5分钟. 空气干燥, 添加20µL 的安装媒体包含 DAPI (请参阅材料表), 用片, 用荧光显微镜 (图 2A) 进行可视化。</li>
	</ol>
	</li>
	<li>对于细胞内抗原的可视化, 重细胞在 PBS 和固定与甲醛 (1% 最终浓度) 为15分钟室温。要 permeabilize 细胞, 添加海卫一 x 100 (0.1%) 和孵化15分钟清洗细胞与2毫升 PBS/0.1% 海卫一 x 100。重在100µL PBS/0.1% 卫一 x 100, 添加抗 vWf 和抗 CD62p 抗体 (1:200 稀释) 和孵化为30分钟室温。<ol>
		<li>洗涤用2毫升 PBS/0.1% 海卫 x 100 和离心机在 400 x g 为 10 min, 重在100µL 同一个缓冲, 并且增加 anti-mouse igg-alexa 594 和兔 igg-alexa 488 (1:100)。在室温下孵育30分钟, 用2毫升 PBS/0.1% 卫重-X 100, 在同一缓冲器的100µL 中冲洗, 并加入20µL 反 CD42b-APC。然后按照步骤3.6.1 的描述将细胞旋转到玻片上, 并为步骤3.6.1 中描述的显微可视化准备样品。</li>
	</ol>
	</li>
	<li>为倍性测定, 收获细胞在天12或13的分化。加1毫升的 SB 洗。离心机在 400 x g 为 10 min 和染色与20µL anti-GPIIb/CD41-FITC 抗体在最后容量100µL. 孵育在4° c 为 30 min。<ol>
		<li>用1毫升的 SB 和重颗粒在300µL 低渗柠檬酸缓冲液 (1.25 毫米柠檬酸钠, 2.5 毫米氯化钠, 3.5 毫米葡萄糖) 中洗涤一次, 含20µg/毫升碘碘化物和0.05% 海卫 x 100。孵育15分钟在4° c 保护免受光。</li>
		<li>添加核糖核酸到最后浓度20µg/毫升和孵化30分钟在4° c 保护免受光。通过收集3万到 5万 CD41-FITC+填充的事件 (图 2C), 通过流式细胞仪确定碘碘化物的强度。</li>
	</ol>
	</li>

4. Proplatelet 计数、血小板计数和血小板活化
<ol>
	<li>收获细胞 (从步骤 3.1) 在8或9天的分化和种子在 1 x 104细胞/良好的 48-井板200µL 的新鲜可持续森林补充与 50 ng/毫升 rhTPO。文化在5天在37° c, 5% CO2. 注: 为定量目的, 种子井三份。这种低密度是需要的可视化和计数 proplatelet 轴承 MK. Proplatelets 通常在2天的文化之后开始出现。峰顶是在天4和5之间。</li>
	<li>使用10X 或20X 目标, 在倒置光显微镜下计算整个井中 proplatelet 轴承 MK 的数量。 注: 一个加热 (37 ° c) 显微镜阶段是可取的, 因为保持在室温下的细胞延长周期导致收缩的 proplatelet 延长。Proplatelets 被观察作为长的引伸从 MK 身体。每个 MK 可能有几个 proplatelet 突起。随着 proplatelets 的发展, MK 体的体积减小。</li>
	<li>收获细胞和离心机在 400 x g 为10分钟在室温。20µL 人 CD41-FITC 抗体的染色细胞, 如步骤3.3 和3.4 所述。计算 proplatelet 轴承 MK (pbMK) 的百分比: pbMK (%) = [(proplatelet-轴承 MKs/井)/(总 CD41+单元格/井)] x 100</li>
	<li>将血小板释放到培养基中, 将细胞与巴斯德吸管轻轻混合, 在14或15的培养基中收集100µL。<ol>
		<li>染色与20µL 人 CD41-FITC 抗体 20-30 分钟在4° c。使用相应的同种控制抗体设置控制管。</li>
		<li>加150µL, 50 µL 数珠。</li>
		<li>对于流细胞分析, 将 FSC 和 SSC 设置为对数刻度。使用 CD41-FITC 中描述的富含血小板的血浆中的正常人血血小板, 如4.4.1 节所述, 为血小板设置浇口 (图 3A)。</li>
		<li>用流式细胞仪分析计数颗粒的染色血小板。收集1000事件的计数珠子使用 FSC 与 SSC 分散图 (图 3A)。使用公式计算基于 CD41-FITC 的正事件 (图 3B) 的血小板数量: 血小板每µL = [(CD41-FITC 阳性事件数)/1000 珠)] x [(50 µL 的珠子数)/样品量)]</li>
	</ol>
	</li>
	<li>为了分析血小板活化, 将细胞与巴斯德吸管轻轻混合, 在14或15的培养基中收集100µL。加入1毫升的 Tyrode 的缓冲液 (137 毫米氯化钠, 2.7 毫米氯化钾, 1 毫米氯化镁2, 1.8 毫米 CaCl2, 0.2 mm Na2HPO4, 12 mm NaHCO3, 5.5 mm d-葡萄糖, pH 6.5) 和离心机在 200 x g 为5分钟的颗粒细胞。<ol>
		<li>收集上清和离心在 800 x g 为10分钟颗粒 platelet-sized 颗粒。</li>
		<li>在 Tyrode 缓冲区的100µL 中丢弃上清和重。添加20µL PAC1-FITC 抗体和腺苷二磷酸 (ADP) 到最后浓度20µM. 室温孵育20分钟, 用流式细胞仪分析确定 FITC 阳性事件的百分比。使用与阳性对照相同的方式处理新鲜的人血小板 (图 3C)。</li>
	</ol>
	</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+continuous+in+vitro+human+platelet+production+using+three-dimensional+scaffolds.">Prolonged continuous in vitro human platelet production using three-dimensional scaffolds.</a> Exp Hematol. 37 (1), 101-110 (2009).</li><li>Proulx, C., Boyer, L., Hurnanen, D. R., Lemieux, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+ex+vivo+expansion+of+megakaryocytes+from+human+cord+blood+CD34+-enriched+cells+in+the+presence+of+thrombopoietin+and+limiting+amounts+of+stem+cell+factor+and+Flt-3+ligand.">Preferential ex vivo expansion of megakaryocytes from human cord blood CD34+-enriched cells in the presence of thrombopoietin and limiting amounts of stem cell factor and Flt-3 ligand.</a> J. Hematother. Stem Cell Res. 12 (2), 179-188 (2003).</li><li>Bornstein, R., Garcia-Vela, J., Gilsanz, F., Auray, C., Cales, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cord+blood+megakaryocytes+do+not+complete+maturation,+as+indicated+by+impaired+establishment+of+endomitosis+and+low+expression+of+G1/S+cyclins+upon+thrombopoietin-induced+differentiation.">Cord blood megakaryocytes do not complete maturation, as indicated by impaired establishment of endomitosis and low expression of G1/S cyclins upon thrombopoietin-induced differentiation.</a> Br. J. Haematol. 114 (2), 458-465 (2001).</li><li>Chong, B. H., Choi, P. Y. I., Khachigian, L., Perdomo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-induced+Immune+Thrombocytopenia.">Drug-induced Immune Thrombocytopenia.</a> Hematol Oncol Clin North Am. 27 (3), (2013).</li><li>Stasi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+thrombocytopenic+purpura:+Current+concepts+in+pathophysiology+and+management.">Idiopathic thrombocytopenic purpura: Current concepts in pathophysiology and management.</a> Thromb Haemost. 99 (1), 4-13 (2008).</li></ol>

作者没有什么可透露的。

本文所述的协议适用于脐血培养中的 MK 和血小板的持续生产。这些细胞可用于研究各种过程, 如药物或生物活动对 MK 增殖、分化、proplatelet 形成和血小板生成的影响。
文中介绍了多种培养基和细胞因子的组合。细胞因子、Flt-3 配体、IL-3 和 IL-6 细胞因子的增加支持 CD34 的增殖.但是, 此扩展在区域性14中导致 MK 纯度降低。这里提出的方法, 使用无血清培养基和 rhTPO 单独, 不允许显着扩展祖细胞, 但允许 unilineage 巨增殖和分化和一致生产 MK (90-99% CD41+CD42a+双阳性细胞) 而不受其他血统的污染。MK 形成的培养期为10至12天, 无需支持基质或饲养细胞。这与需要更大区域性的其他方法 (超过20天)17,18相比比较有利。本议定书的血小板产生率为每位 MK 19-42 血小板, 每输入 CD34 420 血小板, 则为+细胞。大多数协议导致较低的血小板产生率18,19。
虽然产量相对于其他方法高, 但需要大量的 CD34 细胞来产生足够数量的血小板以供治疗使用。脐血 MK 成熟率较低, 一般较小 (低倍性类), 并有降低血小板生产能力的20。然而, 新鲜的联合公司通常是一个更容易获得的资源, 这是一个重要的优势, 为研究员。其他方法, 可以产生更高的产量的 MK 和血小板的治疗目的也被描述13,17。
有一些可变性的来源需要考虑: A) 脐带血单位的质量。仅在24小时内收集的脐带血应用作 CD34 的+细胞的来源。含血块的脐血单位也应丢弃。B) 单个核细胞分数中 CD34 的+单元格的百分比 (步骤 1.8): 使用小于 0.3% CD34+单元格的单核细胞分数可能导致相对较低纯度的 CD34+单元格的生成率较低。这些细胞不推荐用于 MK 分化。重要的是要让液体流失的重力力 (例如, 步骤 1.13, 1.14, 1.16)。在列堵塞的情况下, 建议从磁铁中删除该列, 将电池轻轻地用注射器柱塞插入一个新的管, 并重新加载到一个新的平衡列。
许多条件影响血小板数量和功能。血小板减少指的是明显的下降, 可能导致外部出血和内出血。免疫性血小板减少 (ITP) 和药物引起的血小板减少 (DITP) 是众所周知的导致血小板减少的原因21,22。其他免疫性疾病, 如系统性红斑狼疮和类风湿关节炎也可能对血小板产生有害影响。非免疫性血小板减少的原因包括癌症治疗、严重创伤、感染、骨髓衰竭和手术。由于在化疗或接受干细胞移植的患者中血小板利用率高, 在过去的几十年中, 血小板的输血量稳步增加。MK 和血小板研究无疑将有助于发展大规模的血小板生产, 用于临床应用。可利用的体外产生的功能性血小板可以防止血小板短缺, 并使血小板输注到难治患者。MK 分化和血小板生成体外是研究和理解病理条件和导致血小板形成的生理机制的关键工具。

由于骨髓中的低频率, 在正常的实验室使用中分离出足够数量的初级人类巨 (MK) 是不可行的, 因为它们在0.01% 的有核细胞中占到了1。一种方便的替代方法是在特定生长因子存在的情况下, 对造血干细胞和祖细胞进行体外扩张和分化. 在培养体系中, 有许多细胞因子, 包括干细胞因子、c 型配体和白细胞介素 (IL)-3 和 IL-11 MKs. 生成 (TPO) 是巨文化最有效的生长和分化因子并且是有效的单独或与其他细胞因子, 例如 IL-32。TPO 可以对干细胞种群产生作用, 导致 MKs2的增殖和成熟。
MK 产生血小板从细胞质突起称为 proplatelets 和,在体内,大约 1 x 1011血小板每天形成, 以维持血小板计数 150-400 x 109/l. 血小板生成体外为1000-折叠低于在体内估计3, 这已经产生了许多文化条件使用 CD34+造血祖细胞改善 MK 和血小板生成体外。用于 MK 分化的 CD34+细胞的初始来源是人外周血4。其他细胞来源包括骨髓5,6, 胚胎干细胞/诱导多能干细胞 (ESC/iPSC)7, 脐血 (联合)8,9,10.人骨髓 CD34+ 11和小鼠血统阴性骨髓细胞5 体外产生 MK 和血小板;然而, 缺乏人类骨髓的可用性限制了它作为 CD34 的+细胞的来源。相反, ESC 和 iPSC 代表了体外血小板产生的无限细胞来源。这些细胞的血小板生成需要饲养细胞, 如小鼠 OP9 细胞和较长的培养期。在无馈线条件下获得的血小板似乎不太正常12。iPSC 源性血小板可能在临床上有应用, 因为它们可以扩展到大规模。这个过程需要慢介导转录因子的转导和长期细胞培养13。
CD34 是一个可访问的+单元的来源, 可以在研究设置中随时使用。TPO 单独可以促进脐血衍生 CD34 细胞的分化, 这就产生了高度纯净、成熟的 MKs, 而不需要补充血清或与饲养细胞共培养.其他细胞因子, 如 CD34, 可降低与脐血细胞的分化, 而 Flt-3 配体和 IL-11 促进未成熟巨的产生, 14。本协议描述了从脐血 CD34+细胞在无血清条件下生产高纯度 MK 培养物的情况。

<table>
	<tbody>
		<tr>
			<td>Cell Culture Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human TPO</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-013</td>
			<td></td>
		</tr>
		<tr>
			<td>StemSpan SFEM II</td>
			<td>Stem Cell Technologies</td>
			<td>9605</td>
			<td>Serum-free media for CD34+ cells</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>CD34 Isolation Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 MicroBead kit ultrapure</td>
			<td>Miltenyi Biotec</td>
			<td>130-100-453</td>
			<td>This kit includes the FcR human IgG blocking reagent and CD34 microbeads. These beads contain the anti-CD34 antibody clone QBEND/10. Use a different anti-CD34 clone for purity check (e.g. clone 8G12).</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Alere Technologies</td>
			<td>1114545</td>
			<td>Lymphocyte separation media (density 1.077 g/mL)</td>
		</tr>
		<tr>
			<td>Sterile separation buffer (SB)</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td>This buffer contains phosphate buffered saline (PBS), pH 7.2 containing 0.5% bovine serum albumin and 2 mM EDTA. It can be prepared using sterile, cell culture grade components. De-gas before use because air bubbles can block the column.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Flow Cytometry and Cell Staining Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse anti-Human CD34</td>
			<td>BD Biosciences</td>
			<td>340669</td>
			<td>Clone 8G12. This can be used for CD34 purity check. Final antibody concentration 1:10 dilution.</td>
		</tr>
		<tr>
			<td>PerCP mouse anti-human CD45</td>
			<td>BD Biosciences</td>
			<td>347464</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>PerCP isotype control</td>
			<td>BD Biosciences</td>
			<td>349044</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>FITC Mouse anti-Human CD41a</td>
			<td>BD Biosciences</td>
			<td>340929</td>
			<td>Final antibody concentration 1:5 dilution.</td>
		</tr>
		<tr>
			<td>APC Mouse anti-Human CD42b</td>
			<td>BD Biosciences</td>
			<td>551061</td>
			<td>This antibody can also be used to detect mature MK (the percentage of positive cells in usually lower than with anti CD42a). Final antibody concentration 1:10 dilution.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse anti-Human CD42a</td>
			<td>AbD Serotec</td>
			<td>MCA1227A647T</td>
			<td>Currently distributed by Bio-Rad. Final antibody concentration 1:10 dilution.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse Negative Control</td>
			<td>AbD Serotec</td>
			<td>MCA928A647</td>
			<td>Currently distributed by Bio-Rad. Isotype control antibody</td>
		</tr>
		<tr>
			<td>Anti von Willebrand factor rabbit polyclonal</td>
			<td>Abcam</td>
			<td>AB6994</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>V450 mouse anti-humna CD41a</td>
			<td>BD Biosciences</td>
			<td>58425</td>
			<td>1: 20 dilution</td>
		</tr>
		<tr>
			<td>V450 isotype control</td>
			<td>BD Biosciences</td>
			<td>580373</td>
			<td>1:20 dilution</td>
		</tr>
		<tr>
			<td>PAC1-FITC antibody</td>
			<td>BD Biosciences</td>
			<td>340507</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>Anti CD62p mouse monoclonal</td>
			<td>Abcam</td>
			<td>AB6632</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 goat anti mouse IgG</td>
			<td>Invitrogen</td>
			<td>A11020</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>Ig Isotype Control cocktail-C</td>
			<td>BD Biosciences</td>
			<td>558659</td>
			<td>Isotype control antibody</td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma Aldrich</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr>
			<td>CountBright Absolute Counting Beads</td>
			<td>Molecular Probes, Invitrogen</td>
			<td>C36950</td>
			<td>Counting beads</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>Smaller and larger columns are also commercially available</td>
		</tr>
		<tr>
			<td>MidiMACS Separator magnet</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 5mL round bottom polypropylene FACS tubes, with Snap Cap, Sterile</td>
			<td>In Vitro technologies</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Menzel-Glaser</td>
			<td>J3800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>Antifade mounting medium with DAPI</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Leica</td>
			<td>DMIRB inverted microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Zeiss</td>
			<td>Vert.A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell analyser</td>
			<td>BD Biosciences</td>
			<td>FACS Canto II</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytospin centrifuge</td>
			<td>ThermoScientific</td>
			<td>Cytospin 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell analyser software</td>
			<td>BD Biosciences</td>
			<td>FACS Diva Software</td>
			<td></td>
		</tr>
		<tr>
			<td>Single cell analysis software</td>
			<td>Tree Star</td>
			<td>FlowJo</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope software</td>
			<td>Zeiss</td>
			<td>Zen 2 blue edition</td>
			<td></td>
		</tr>
	</tbody>
</table>

megakaryocyte differentiation and platelet formation from human cord blood-derived cd34+ cells

血小板的产生主要发生在骨髓的过程中, 称为形成。在形成, 造血祖细胞分化形成血小板前体称为巨, 后者的末期分化, 释放血小板从长细胞质过程称为 proplatelets。巨是罕见的细胞局限于骨髓, 因此很难获得足够数量的实验室使用。在合适的条件下, 通过培养 CD34 的+细胞, 可以在体外实现高效的人巨生产。这里详述的协议描述了由脐带血标本中的磁性细胞分类分离 CD34 的+细胞。介绍了在无血清条件下生产高纯度、成熟巨的必要步骤。还提供了巨分化的表型分析和 proplatelet 形成和血小板生成的测定的详细资料。影响巨分化和/或 proplatelet 形成的效应, 如血小板抗体或生成拟, 可以添加到培养细胞, 以检查生物功能。

此协议允许从脐血衍生的 CD34+细胞制备高度纯净的 MK 培养物。脐血中 CD34+单元格的百分比约为 1.3%15 (图 1A), 每个单核细胞的总数量 (步骤 1.8) 范围从 90-300 x 106每个单元。隔离后的 CD34+/CD45+ 细胞的纯度从90到 99% (图 1B)。MK (定义为 CD41 的+单元格) 在无血清 CD34+单元格区域性中早期观察到 rhTPO 的存在。在7天, 成熟的 MK (CD41+和 CD42a+单元格) 的百分比通常为 30-40% (图 1C)。最高级别的 CD41+和 CD42a+双阳性单元格 (90-99%) 在天10和12之间被观察到差异 (图 1C)。观察到的变异性主要取决于脐带血源和在步骤1.17 中隔离的 CD34+/CD45+单元的纯度。10天成熟 MK 的收率范围从 5-10 每输入 CD34+单元格不等。在荧光显微镜下观察到的成熟 MK (CD41+/CD42b+) 显示在图 2A中。培养的 MK 显示为大的, 通常核的细胞 (图 2A, 箭头)。MK 的颗粒含量由 vWf (绿色) 和 CD62p (p-选择素, 红色) 染色 (图 2B) 决定。在培养的 MKs 中观察到的倍性分布见图 2C。
Proplatelets 是长, 串珠纤维或花丝, 从 MK 身体延伸。Proplatelets 可以是几百微米长的16并且包含分支和肿胀区域。在图 2D中说明了具有 proplatelet 特性的 MK。proplatelet 轴承 MK (pbMK) 的百分比为1.3 ±0.17%。
血小板可以按照协议中的描述进行分析和计算。如图 3B所示, 细胞培养上清液中的大多数血小板都属于外周血血小板分析门, 对血小板标记 CD41 (图 3B) 是阳性的。该方法的血小板产率为每 MK 19 至42血小板不等. 在培养的血小板激动剂, 如 ADP, 由活化特异性 PAC1 单克隆抗体的增强结合所决定, 可活化血小板激活 (图 3C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56420/56420fig1.jpg"> 
图 1: CD34 的流式细胞仪图形+细胞分离和 MK 分化的培养.(A) 从人脐血 (步骤 1.8) 中纯化的单核细胞 (左面板显示的门控) 用 anti-CD34-PE 抗体染色, 以确定样本中 CD34+单元格的百分比 (1.3%, 右面板)。(B)分离后, 阳性分数 (步骤 1.17) 用 anti-CD34-PE 和 anti-CD45 PerCP 抗体染色。图 (98.1%、右面板、右上象限) 中显示了丰富的 CD34+填充。(C) 在7天或11天内, CD34+ 细胞对 MK 分化的体外进行表型分析, anti-CD41 和 anti-CD42a 抗体染色。成熟的 MK 对 CD41+和 CD42a+ (右上象限) 都是正的。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56420/56420fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56420/56420fig2.jpg"> 
图 2: MK 染色、倍性和 proplatelet 形成体外.(A) 11 天 MK 的荧光图像, anti-CD41-PE 和 CD42b-APC 抗体染色。细胞核被 DAPI 染色。黄色箭头表示 multi-nuclear MKs. 缩放栏, 30 µm. (B) 荧光图像14天 MK 染色, 反 vWf (绿色), 抗 CD62p (红色) 和 anti-CD42b-APC (洋红) 抗体。细胞核被 DAPI 染色。缩放条, 15 µm (C) 具有代表性的门控策略, 显示 CD41+事件的倍性分布。图 (下面板) 显示了被观察到的倍性类的分布 (n = 4), 误差条, SD. (D) 显示了 proplatelet 轴承 MK 的特征形态。MK 体由箭头指示。从 MK (proplatelets) 延伸的长胞质过程由箭头表示。在某些领域或视野中, 是否有一两个 MK 正在生产这些 proplatelet 的扩展, 可能还不清楚。这应该算作一个 proplatelet 的 MK. 用倒置显微镜拍摄的图像, 10X 目标。标尺, 50 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56420/56420fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56420/56420fig3.jpg"> 
图 3: 大量的血小板产生了体外和血小板活化.(A) 用富含血小板的血浆中的人血小板来设置分析门, 使用对数刻度进行前向和侧向散射 (左面板)。右面板显示来自 MK 区域性的单元格。血小板产生的体外在为人类血小板定义的分析门中观察到。图中显示了细胞和计数珠子。血小板 (B)在体外产生的血小板 (anti-CD41 抗体) 染色。利用血小板富集等离子体中的人血小板, 用对数标度对前、侧散射进行分析, 并比较 CD41-FITC 门内的剖面。FITC, FITC 同种控制(c)经 ADP 治疗后的血小板活化, 最终浓度为20µM. 用血小板丰富的血浆中的血小板作为对照 (上部板)。在培养基中产生的血小板在下部板中显示。PAC1 抗体的结合表明血小板活化。C-FITC, FITC 同种控制。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56420/56420fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells at JoVE.com

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

高纯度的巨可以从脐带血衍生的 CD34+细胞获得。本文介绍了一种 CD34+单元格隔离和巨区分的方法。

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 ...

megakaryocyte-differentiation-platelet-formation-from-human-cord

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

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

19.5K 次观看.  University of New South Wales. 高纯度的巨可以从脐带血衍生的 CD34+细胞获得。本文介绍了一种 CD34+单元格隔离和巨区分的方法。

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

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

科研

免疫与感染

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 ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

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

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

从人类胸腺髓样树突状细胞和上皮细胞的分离

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

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

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

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

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

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

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

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

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

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

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

在从幼稚的CD4 + T细胞的人CD4 + FOXP3 +诱导调节性T细胞（iTregs的）使用含有TGF-β-协议的体外分化

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

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.

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 ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

人网膜和皮下脂肪组织 CD34+CD31+ 内皮细胞的分离、扩增和脂肪诱导

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

人脐血巨分化与血小板形成的 CD34⁺单元格

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