我们感谢露西亚 Sammarco 和她的露露的柠檬水站的灵感, 动力, 和支持。这项工作得到了部分由 NIH R01 AI102893 和 R01 CA179363 (克里希纳) 的支持;NHLBI-HL087951 (纳丹);NIH-CA151893-K08 (M.J.R.);1R01CA164225 (L. W);亚历克斯柠檬水站立基础 (克里希纳);委员会基金的 HRHM 计划 (克里希纳;纳丹;硕士 T);尼古拉斯家族基金会 (克里希纳);Gardetto 家族 (克里希纳);现代学者计划 (M.S.T.);现代希望在轮子 (纳丹);委员会基金 (M.S.T. 和克里希纳);儿童研究所, MCW (纳丹);凯西菲 Fogerty 奖 (M.J.R.)。

所有动物协议跟随了动物的人道和道德治疗并且由机构动物护理和用途委员会 (IACUC) 批准在威斯康辛医学学院的生物医学研究中心 (MCW), 密尔沃基, WI。使用人外周血单个核细胞 (PBMCs) 被批准的机构审查委员会 (IRB) 血液研究所的血液研究中心威斯康星州, 密尔沃基, WI。
1. 小鼠、细胞系和载体
<ol><li>从商业供应商那里获得 C57BL/6 的老鼠。保持在无病原体条件下的小鼠菌落, 并在6和12周的年龄段使用雌性和雄性小鼠。从 IRB 批准的来源获得去除人 PBMCs。</li>
	<li>麻醉在丙烯乙二醇 (1, 2-丙二醇, USP 等级) 中混合 20-30% v/v 异氟醚的动物, 以麻醉小鼠。将兽医软膏涂抹于眼部, 防止小鼠麻醉时干燥。</li>
	<li>对动物进行安乐, 在麻醉诱导后进行颈椎脱位。<ol>
<li>用一只手牢牢抓住尾巴的底座来抑制老鼠。在头骨的底部放置一支结实的棍子型钢笔或拇指和另一只手的手指, 放在颈部的后部。</li>
		<li>为了产生脱位, 推手抑制动物的头部向前, 并向下推, 而向后拉的手持有的尾巴基地。通过感觉宫颈组织分离来验证脱位的有效性。</li>
	</ol>
</li>
	<li>保持在室内/笼子里的动物至少5分钟, 并删除他们只有当呼吸活动是缺席或当有一个检测的心跳缺乏。</li>
	<li>从商业供应商那里获得 K562 和 YAC-1 细胞, 并在含有10% 热灭活 FBS 的 RPMI1640 培养基中维持它们。定期测试这些细胞系, 以排除支原体污染的可能性。</li>
</ol>2. 慢载体的制备和滴定
<ol><li>培养 5 x 106 293T 细胞过夜在 T75 cm2烧瓶中, 含有20毫升的 RPMI1640 培养基, 10% FBS, 100 U/毫升青霉素, 100 µg/毫升链霉素, 1 毫米丙酮酸钠, 5% 的7.5% 碳酸氢钠溶液, 0.001%β-基。将烧瓶放入37° c 的恒温箱中, 注入 5.2% CO2。</li>
	<li>用磷酸缓冲盐 (PBS) 中的胰蛋白酶 (0.025%)/EDTA (1 毫米) 获得293T 细胞。在 PBS 中加入5毫升的胰蛋白酶/EDTA, 在37° c 的孵化箱中孵育10分钟的烧瓶, 使293T 细胞脱离 T75 cm2烧瓶。<ol>
<li>收集分离的细胞, 并清洗两次在 PBS, 以消除任何痕迹的胰蛋白酶和 EDTA。用例计数单元格, 并将单元格数调整为每毫升100万单元格。</li>
	</ol>
</li>
	<li>染的293T 细胞18与3.95 µg 的 psPAX2, 1.32 µg pMD2G, 和5.26 µg pLEP-gfp 普洛 (生成 in-house 在 EF1alpha 的克隆 pWPI 启动子, 在 pLenti 的地方的巨细胞病毒的启动子,-gfp-普洛) 19.<ol>
<li>准备1毫升150毫米氯化钠加63µL 0.6 毫克/毫升亚 (PEI; 2.5万 kD, 线形)。混合好, 然后加入质粒和混合。在室温下孵育20分钟, 然后将其添加到细胞中。</li>
		<li>16 h post-transfection, 用新鲜培养基加4.5 毫米丁酸钠取代转染培养基。收获含有病毒 48 h post-transfection 的上清液, 用隔夜离心法浓缩 5000 x g。请参阅材料表。</li>
	</ol>
</li>
	<li>通过流式细胞仪 72 h post-transduction20进行串行稀释和检测, 确定使用293T 细胞的病毒滴。使用绿色荧光蛋白 (GFP) 的表达作为量化病毒滴的措施。</li>
</ol>3. 小鼠原代 NK 细胞的纯化和扩展
<ol><li>纯化小鼠原发性 NK 细胞21。简要地, 通过尼龙羊毛柱通过单细胞脾悬浮物, 以耗尽由 B 细胞和巨细胞组成的黏附人群。</li>
	<li>培养换药种群洗从尼龙羊毛柱, 其中含有 1000 u/毫升白细胞介素 (IL)-2 在 RPMI1640 培养基与 10% FBS, 100 U/毫升青霉素, 100 µg/毫升链霉素, 1 毫米丙酮酸钠, 5% 7.5% 碳酸氢钠溶液, 和0.001% β-基 (RPMI1640 完全介质)。</li>
	<li>改变这种培养基4天的烧瓶, 以消除换药 T 和 NKT 细胞;仍然附着在烧瓶中的细胞主要是 NK 细胞。添加20毫升新鲜 RPMI1640 完全培养基和 1000 U/毫升 IL-2, 以补充烧瓶。</li>
	<li>在7天通过流式细胞仪检测 nk 细胞培养物的纯度, 并使用具有95% 以上 CD3 -NK1.1+填充的准备工作。</li>
</ol>4. 人类原发性 NK 细胞的纯化与扩展
<ol><li>通过密度梯度分离 PBMCs, 从健康的人类志愿者的巴菲大衣。简要地, 仔细地35毫升的 half-diluted (与 HBSS) 细胞超过15毫升的密度梯度在一个50毫升规范管。离心机在 400 x g 为30分钟在20° c 在摇摆的桶转子没有闸。</li>
	<li>根据制造商的协议, 使用商用抗体阴性选择套件来纯化人类原代 NK 细胞。</li>
	<li>分离后, 通过免疫荧光分析测定 NK 细胞的纯度。用2µg/毫升 anti-CD3 和2µg/毫升 anti-CD56 抗体在4° c 下染色 nk 细胞, 20 min. 在细胞表面没有 CD3 和 CD56 存在的情况下, 用 PBS nk 细胞的数量来冲洗两次细胞。</li>
	<li>用抗体在4° c 下孵育20分钟的细胞制剂, 用 PBS 进行冲洗, 并在流式仪中进行分析。使用 NK 细胞准备与超过85% 纯度的基因转导化验20。</li>
</ol>5. 小鼠和人原发性 NK 细胞与慢的转导
<ol><li>在 0.5 x 105/毫升培养基中, 在有 GFP 慢上清的情况下, 在5处将小鼠或人类主 NK 细胞悬浮在24井板上, 10、20种感染 (莫伊) 和有 Pb (8 µg/毫升)、PS (8 µg/毫升) 或葡聚糖 (8 µg/毫升) 的存在。</li>
	<li>离心板在 1000 x g 为60分钟。</li>
	<li>没有迁上清液, 培养细胞过夜 (16-18 小时) 在37° c 孵化器注入 5.2% CO2.
</li>
	<li>在 IL-2 (300 U/毫升) 的情况下, 用10毫升 PBS 和重在2毫升完全 RPMI1640 培养基中洗涤。</li>
	<li>分别通过在不同的 effector-to-target 细胞比22中执行51铬 (Cr) 释放试验, 测试人和小鼠主要 NK 细胞对 K562 和 YAC-1 的杀伤作用。<ol>
<li>简要地, 给100万靶细胞50µCi 标记钠铬酸盐51Cr。在4小时的潜伏期, 他们吸收了51Cr 为细胞蛋白。在孵化结束时, 清洗细胞以移除任何未注册的标签。</li>
		<li>通过从目标细胞中的绝对、自发和实验释放的51Cr 的数量来计算特定肿瘤细胞的裂解。 注: pentaplicate 实验的具体溶解百分率的计算采用以下公式:% 特定的裂解 = ((51cr 平均实验释放- 51cr 平均自发释放)/(51cr 平均值最大释放- 51cr 平均自发释放)) х 100, 其中 "51cr 平均自发释放" 是51cr 释放的目标细胞在缺乏 NK 细胞和 "51cr 平均最大释放" 是51cr由2的盐酸 (HCl) 溶解后从靶细胞释放出来。</li>
	</ol>
</li>
	<li>在7天转 NK 细胞, 轻轻敲击烧瓶。<ol>
<li>通过涂层 96-好, 高蛋白吸收聚苯乙烯板, 2.5 µg/毫升浓度的 anti-NKG2D 单克隆在一夜之间激活滴定浓度的板结合 anti-NKG2D (A10) 抗体的收获细胞。</li>
		<li>在原代 NK 细胞加入前, 用100µL PBS 三次冲洗每一个井。</li>
	</ol>
</li>
	<li>收集文化清使用多通道吸管在16和 18 h 后之间定量细胞因子例如干扰素γ。使用与酶联免疫吸附试验 (ELISA) 试剂盒提供的重组细胞因子生成标准曲线。</li>
	<li>用 annexin-V/7-amino-actinomycin D (7-反倾销) 染色法测定 nk 细胞的存活率23 , 并利用流式细胞仪测定转化 nk 淋巴细胞中坏死细胞的百分比。<ol>
<li>为了进行这种化验, 在转导后四天内采集小鼠和人 NK 细胞, 用10毫升的冷 PBS 在 500 x g 分别洗涤两次, 每5分钟, 用蛋白 V (PE)/7-反倾销试剂盒孵育。</li>
	</ol>
</li>
	<li>使用适当的流式细胞仪软件对数据进行分析。选择活细胞种群并分析 GFP (FITC 通道) 的表达作为病毒转导的一种措施24。</li>
</ol>

<ol>
	<li>Vivier, E., Tomasello, E., Baratin, M., Walzer, T., Ugolini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+natural+killer+cells.">Functions of natural killer cells.</a> Nat. Immunol. 9, 503-510 (2008).</li><li>Zitvogel, L., Tesniere, A., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+despite+immunosurveillance:+immunoselection+and+immunosubversion.">Cancer despite immunosurveillance: immunoselection and immunosubversion.</a> Nat. Rev. Immunol. 6, 715-727 (2006).</li><li>Arina, A., 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=Cellular+liaisons+of+natural+killer+lymphocytes+in+immunology+and+immunotherapy+of+cancer.">Cellular liaisons of natural killer lymphocytes in immunology and immunotherapy of cancer.</a> Expert. Opin. Biol. Ther. 7, 599-615 (2007).</li><li>Manilay, J. O., Sykes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+killer+cells+and+their+role+in+graft+rejection.">Natural killer cells and their role in graft rejection.</a> Curr. Opin. Immunol. 10, 532-538 (1998).</li><li>Raulet, D. H., Vance, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-tolerance+of+natural+killer+cells.">Self-tolerance of natural killer cells.</a> Nat. Rev. Immunol. 6, 520-531 (2006).</li><li>Chouaib, S., 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=Improving+the+outcome+of+leukemia+by+natural+killer+cell-based+immunotherapeutic+strategies.">Improving the outcome of leukemia by natural killer cell-based immunotherapeutic strategies.</a> Front Immunol. 5, 95 (2014).</li><li>Dulphy, N., 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=Underground+Adaptation+to+a+Hostile+Environment:+Acute+Myeloid+Leukemia+vs.+Natural+Killer+Cells.">Underground Adaptation to a Hostile Environment: Acute Myeloid Leukemia vs. Natural Killer Cells.</a> Front Immunol. 7, 94 (2016).</li><li>Tran, J., Kung, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vectors+mediate+stable+and+efficient+gene+delivery+into+primary+murine+natural+killer+cells.">Lentiviral vectors mediate stable and efficient gene delivery into primary murine natural killer cells.</a> Mol. Ther. 15, 1331-1339 (2007).</li><li>Maasho, K., Marusina, A., Reynolds, N. M., Coligan, J. E., Borrego, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+human+natural+killer+cell+line,+NKL,+using+the+Amaxa+nucleofection+system.">Efficient gene transfer into the human natural killer cell line, NKL, using the Amaxa nucleofection system.</a> J. Immunol. Methods. 284, 133-140 (2004).</li><li>Trompeter, H. I., Weinhold, S., Thiel, C., Wernet, P., Uhrberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+highly+efficient+gene+transfer+into+natural+killer+cells+by+nucleofection.">Rapid and highly efficient gene transfer into natural killer cells by nucleofection.</a> J. Immunol. Methods. 274, 245-256 (2003).</li><li>Becknell, B., 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=Efficient+infection+of+human+natural+killer+cells+with+an+EBV/retroviral+hybrid+vector.">Efficient infection of human natural killer cells with an EBV/retroviral hybrid vector.</a> J. Immunol. Methods. 296, 115-123 (2005).</li><li>Jiang, K., 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=Syk+regulation+of+phosphoinositide+3-kinase-dependent+NK+cell+function.">Syk regulation of phosphoinositide 3-kinase-dependent NK cell function.</a> J. Immunol. 168, 3155-3164 (2002).</li><li>Burshtyn, D. N., 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=Conserved+residues+amino-terminal+of+cytoplasmic+tyrosines+contribute+to+the+SHP-1-mediated+inhibitory+function+of+killer+cell+Ig-like+receptors.">Conserved residues amino-terminal of cytoplasmic tyrosines contribute to the SHP-1-mediated inhibitory function of killer cell Ig-like receptors.</a> J. Immunol. 162, 897-902 (1999).</li><li>Schroers, 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=Gene+transfer+into+human+T+lymphocytes+and+natural+killer+cells+by+Ad5/F35+chimeric+adenoviral+vectors.">Gene transfer into human T lymphocytes and natural killer cells by Ad5/F35 chimeric adenoviral vectors.</a> Exp. Hematol. 32, 536-546 (2004).</li><li>Imai, C., Iwamoto, S., Campana, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+modification+of+primary+natural+killer+cells+overcomes+inhibitory+signals+and+induces+specific+killing+of+leukemic+cells.">Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells.</a> Blood. 106, 376-383 (2005).</li><li>Denning, W., 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=Optimization+of+the+transductional+efficiency+of+lentiviral+vectors:+effect+of+sera+and+polycations.">Optimization of the transductional efficiency of lentiviral vectors: effect of sera and polycations.</a> Mol. Biotechnol. 53, 308-314 (2013).</li><li>Lamers, C. H., Willemsen, R. A., Luider, B. A., Debets, R., Bolhuis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+gene+transduction+and+expansion+of+human+T+lymphocytes+for+clinical+immunogene+therapy+of+cancer.">Protocol for gene transduction and expansion of human T lymphocytes for clinical immunogene therapy of cancer.</a> Cancer Gene Ther. 9, 613-623 (2002).</li><li>Campeau, E., 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=A+versatile+viral+system+for+expression+and+depletion+of+proteins+in+mammalian+cells.">A versatile viral system for expression and depletion of proteins in mammalian cells.</a> PLoS. ONE. 4, e6529 (2009).</li><li>Segura, M. M., Garnier, A., Durocher, Y., Ansorge, S., Kamen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+protocol+for+lentiviral+vector+mass+production.">New protocol for lentiviral vector mass production.</a> Methods Mol. Biol. 614, 39-52 (2010).</li><li>Rajasekaran, K., 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=Signaling+by+Fyn-ADAP+via+the+Carma1-Bcl-10-MAP3K7+signalosome+exclusively+regulates+inflammatory+cytokine+production+in+NK+cells.">Signaling by Fyn-ADAP via the Carma1-Bcl-10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells.</a> Nat. Immunol. 14, 1127-1136 (2013).</li><li>Regunathan, J., Chen, Y., Wang, D., Malarkannan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKG2D+receptor-mediated+NK+cell+function+is+regulated+by+inhibitory+Ly49+receptors.">NKG2D receptor-mediated NK cell function is regulated by inhibitory Ly49 receptors.</a> Blood. 105, 233-240 (2005).</li><li>Awasthi, A., 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=Rap1b+facilitates+NK+cell+functions+via+IQGAP1-mediated+signalosomes.">Rap1b facilitates NK cell functions via IQGAP1-mediated signalosomes.</a> J. Exp. Med. 207, 1923-1938 (2010).</li><li>Rajasekaran, K., 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=Transforming+Growth+Factor-{beta}-activated+Kinase+1+Regulates+Natural+Killer+Cell-mediated+Cytotoxicity+and+Cytokine+Production.">Transforming Growth Factor-{beta}-activated Kinase 1 Regulates Natural Killer Cell-mediated Cytotoxicity and Cytokine Production.</a> J Biol. Chem. 286, 31213-31224 (2011).</li><li>Wolkowicz, R., Nolan, G. P., Curran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vectors+for+the+delivery+of+DNA+into+mammalian+cells.">Lentiviral vectors for the delivery of DNA into mammalian cells.</a> Methods Mol. Biol. 246, 391-411 (2004).</li><li>Sutlu, T., 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=Inhibition+of+intracellular+antiviral+defense+mechanisms+augments+lentiviral+transduction+of+human+natural+killer+cells:+implications+for+gene+therapy.">Inhibition of intracellular antiviral defense mechanisms augments lentiviral transduction of human natural killer cells: implications for gene therapy.</a> Hum. Gene Ther. 23, 1090-1100 (2012).</li><li>Cornetta, K., Anderson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protamine+sulfate+as+an+effective+alternative+to+polybrene+in+retroviral-mediated+gene-transfer:+implications+for+human+gene+therapy.">Protamine sulfate as an effective alternative to polybrene in retroviral-mediated gene-transfer: implications for human gene therapy.</a> J. Virol. Methods. 23, 187-194 (1989).</li><li>Toyoshima, K., Vogt, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+and+inhibition+of+avian+sarcoma+viruses+by+polycations+and+polyanions.">Enhancement and inhibition of avian sarcoma viruses by polycations and polyanions.</a> Virology. 38, 414-426 (1969).</li><li>Jensen, M., 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=The+bi-specific+CD3+x+NCAM+antibody:+a+model+to+preactivate+T+cells+prior+to+tumour+cell+lysis.">The bi-specific CD3 x NCAM antibody: a model to preactivate T cells prior to tumour cell lysis.</a> Clin. Exp. Immunol. 134, 253-263 (2003).</li></ol>

提交人声称没有任何经济利益冲突。

本研究表明, 用葡聚糖作为阳离子聚合物剂, 可提高小鼠和人原代 NK 细胞的慢转导效率。此外, 其他阳离子剂, 如 Pb 或 PS, 对病毒载体向人类原发性 NK 细胞的传递无明显影响。在此之前, 人们已经证明, Pb 可以增强人类 T 细胞的基因转导17。然而, 这些结果表明, 无论是 Pb 还是 PS 在人类原代 NK 细胞上都没有类似的效率。在本研究中, Pb 对小鼠 NK 细胞的转导效果仅有轻度改善。结果?...

自然杀手 (NK) 细胞是先天免疫系统的主要淋巴细胞种群1。NK 细胞作为宿主免疫应答的一线防御者, 抗肿瘤和感染2,3,4。NK 细胞通过分泌强有力的细胞因子和趋化因子5在耐受性发展中起着重要作用。由于其靶向和消除肿瘤细胞的强大能力, 目前正在进行多项临床试验, 以评估供者派生的人类 NK 细胞作为癌症的过继免疫治疗6,7。与 T 细胞相比, NK 细胞的发育生物学尚未良好8。这种缺乏知识的部分原因是缺少有效的技术来提供老鼠或人类原发性 NK 细胞感兴趣的基因。由于这些原因, 大多数 NK 细胞的研究都是在细胞系中进行的, 而不是在原代细胞中进行。因此, 需要一个可靠和有效的协议, 传感器原代 NK 细胞与感兴趣的基因是至关重要的。
这项研究的总目标是制定一个一致的和可靠的方法, 其中主要人或小鼠 NK 细胞可以转与 lenti 或病毒。
早先的研究试图解决这个问题, 主要是利用了原代 NK 细胞的瞬时转化。这包括质粒转染9,10, eb 病毒-巴尔毒 (eb)/逆转录酶杂交载体11, 痘苗向量12,13, 和 Ad5/F35 嵌合体腺病毒载体14。尽管这些技术的效率很高, 但转导的瞬态性使得它们不适合长期利用转基因 NK 细胞。最近的一些研究已经使用逆转录病毒载体传感器 NK 细胞, 需要多个感染周期来达到一个可接受的基因表达水平11,15。与逆转录病毒载体不同, 慢载体可以利用宿主细胞核进口机械转移病毒 pre-integration 复合体进入细胞核。这是在 non-dividing 细胞 (包括原代 NK 细胞) 中复制病毒的一个主要限制因素。
不同的细胞表面受体和病毒粒子之间的相互作用允许病毒对细胞的吸收。病毒包膜蛋白及其同源宿主受体之间的初始接触可能会受到限制, 因为这两者之间存在潜在的负电荷。许多转导技术的基本原理是, 添加阳离子聚合物, 如凝聚 (Pb), 硫酸精蛋白 (PS), 或葡聚糖, 可以给细胞表面受体积极的电荷, 从而增强病毒包络的结合蛋白质.这将提高融合效率和吸收的病毒粒子的细胞16。虽然据报道, Pb 或 PS 可以改善 T 细胞的基因转移17, 他们的应用没有任何影响的转导效率的原代 NK 细胞。此外, 还没有对这些试剂使用原代 NK 细胞进行比较分析。本研究对三阳离子聚合物的转导效率进行了比较。结果表明, 在这三阳离子聚合物中, 只有葡聚糖能显著提高小鼠和人原代 NK 细胞的有效病毒转导。

<table>
	<tbody>
		<tr>
			<td>Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>90-64-91-9</td>
			<td></td>
		</tr>
		<tr>
			<td>polybrene (Pb)</td>
			<td>Sigma-Aldrich</td>
			<td>TR-1003</td>
			<td></td>
		</tr>
		<tr>
			<td>protamine sulfate (PS)</td>
			<td>Sigma-Aldrich</td>
			<td>p3369</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Corning</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Corning</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ATALANTA</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Corning</td>
			<td>30-001-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>B-mercaptoethanol</td>
			<td>SIGMA</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium pyruvate</td>
			<td>Corning</td>
			<td>MT25000CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Interferon gamma (IFN-&#947; )</td>
			<td>eBioscience</td>
			<td>14-7311-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium lodding staining solution</td>
			<td>BD</td>
			<td>51-66211E</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000</td>
			<td>Thermo Fisher</td>
			<td>L3000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>PHOENIX</td>
			<td>NDC 57319-559-05</td>
			<td></td>
		</tr>
		<tr>
			<td>NK cell negative selection kit</td>
			<td>Stem Cell</td>
			<td>19855</td>
			<td></td>
		</tr>
		<tr>
			<td>Yac-1</td>
			<td>ATCC</td>
			<td>TIB-160</td>
			<td></td>
		</tr>
		<tr>
			<td>K562</td>
			<td>ATCC</td>
			<td>CCL-243</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Jakson</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flasks</td>
			<td>Cornnig</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>antibody-based negative selection kits</td>
			<td>Stem Cell</td>
			<td>19055</td>
			<td></td>
		</tr>
		<tr>
			<td>51Chromium (Cr)-release assays</td>
			<td>perkin elmer&#39;s</td>
			<td>NEZ030</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA kits</td>
			<td>Ebioscience</td>
			<td>00-4201-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Butyrate</td>
			<td>Sigma</td>
			<td>5887-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear polyethylenimine</td>
			<td>polysciences</td>
			<td>23966-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>GE Life Science</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Corning</td>
			<td>21-022-CV</td>
			<td></td>
		</tr>
	</tbody>
</table>

dextran enhances the lentiviral transduction efficiency of murine and human primary nk cells

将特定基因的有效转导转化为自然杀伤细胞是一个主要的挑战。成功的 transductions 是关键的定义的作用基因的兴趣在发展, 分化和功能的 NK 细胞。最近的进展与嵌合抗原受体 (汽车) 的癌症免疫治疗强调需要一个有效的方法提供外源基因的效应淋巴细胞。慢介导的基因 transductions 对原人或小鼠 NK 细胞的效率仍然很低, 这是一个主要的限制因素。最近的进展, 使用阳离子聚合物, 如凝聚, 显示了改善基因转导效率的 T 细胞。然而, 这些产品未能改善 NK 细胞的转导效率。这项研究表明, 葡聚糖是一种支链多糖, 能显著提高人和小鼠原代 NK 细胞的转导效率。这种高度可再生的转导方法为换人类原发性 nk 细胞提供了一个有效的工具, 它可以极大地改善临床基因的传递应用, 从而使 nk 干细胞免疫治疗.

葡聚糖诱导人和小鼠 NK 细胞慢载体的高效基因转移
人类 NK 细胞从 PBMC 分离纯化 (纯度超过 85%), 并在夜间与 rIL-2 300 U/毫升孵育。这些主要 NK 细胞, 然后转与 GFP 慢在不同的多重感染 (莫伊; 3, 10, 20 每细胞) 24-井板存在8µg/毫升铅, PS, 或葡聚糖。细胞被离心在 1000 x g 为60分钟和培养 (在病毒面前) 过夜在37° c 在 CO2孵化器。细?...

Watch this Scientific Journal Video about Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells at JoVE.com

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

这项研究的目的是制定技术, 允许成功的基因转导在原自然杀手 (NK) 细胞。葡聚糖介导的人或小鼠原代 NK 细胞的慢转导可导致较高的基因表达效率。这种基因转导方法将极大地改善 NK 细胞的遗传操作。

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 ...

dextran-enhances-lentiviral-transduction-efficiency-murine-human

Research

JoVE Journal

Immunology and Infection

10.9K 次观看.  The Blood Center of Wisconsin. 这项研究的目的是制定技术, 允许成功的基因转导在原自然杀手 (NK) 细胞。葡聚糖介导的人或小鼠原代 NK 细胞的慢转导可导致较高的基因表达效率。这种基因转导方法将极大地改善 NK 细胞的遗传操作。

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

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

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

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

人外周血NK细胞的扩展，纯化和功能评估

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

科研

免疫与感染

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 ...

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 ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

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, ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

人外周血和淋巴组织中原发性 T 细胞突触界面的评价

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

利用 Cas9 Ribonucleoproteins 生成人 NK 细胞的研究

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

原代人细胞中寨卡病毒抗体依赖性增强的定量研究

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...