Name: isolation of leukocytes from human breast milk for use in an antibody-dependent cellular phagocytosis assay of hiv targets
Uploaded: 2019-09-06T14:00:00
Description: Watch this Scientific Journal Video about Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets at JoVE.com

我们感谢西奈山伊坎医学院医学系和微生物学系的苏珊·佐拉-帕斯纳博士的手稿评论。NIH/NICHD为该项目提供了资助，赠款号为R21 HD095772-01A1。此外，R. Powell还得到西奈山伊坎医学院传染病司医学部的资金支持。

本研究的每个参与者都是根据道德和机构审查委员会 （IRB） 的批准招募和面试的，并经过西奈山人类主体保护计划 （PPHS） 的指导和授权，使用 IRB 批准的获取母乳样本的协议。
1. 目标微球制备
<ol><li>选择相关靶抗原。 注：在此示例中，使用了重组蛋白V1V2-2F5K，它旨在模仿原生HIV包络30的三分位。</li>
	<li>根据制造商的协议，?...

<ol>
	<li>Hassiotou, F., Geddes, D. T., Hartmann, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+in+human+milk:+state+of+the+science.">Cells in human milk: state of the science.</a> Journal of Human Lactation. 29 (2), 171-182 (2013).</li><li>Lonnerdal, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+and+physiologic+significance+of+human+milk+proteins.">Nutritional and physiologic significance of human milk proteins.</a> The American Journal of Clinical Nutrition. 77 (6), 1537S-1543S (2003).</li><li>Butte, N. F., Garza, C., Stuff, J. E., Smith, E. O., Nichols, B. 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Effect of breastfeeding on infant and child mortality due to infectious diseases in less developed countries: a pooled analysis.</a> Lancet. 355 (9202), 451-455 (2000).</li><li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Updates+on+HIV+and+Infant+Feeding:+The+Duration+of+Breastfeeding,+and+Support+from+Health+Services+to+Improve+Feeding+Practices+Among+Mothers+Living+with+HIVWHO+Guidelines+Approved+by+the+Guidelines+Review+Committee.">Updates on HIV and Infant Feeding: The Duration of Breastfeeding, and Support from Health Services to Improve Feeding Practices Among Mothers Living with HIVWHO Guidelines Approved by the Guidelines Review Committee.</a> World Health Organization. , (2016).</li><li>Nelson, C. 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G., 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=HIV-specific+functional+antibody+responses+in+breast+milk+mirror+those+in+plasma+and+are+primarily+mediated+by+IgG+antibodies.">HIV-specific functional antibody responses in breast milk mirror those in plasma and are primarily mediated by IgG antibodies.</a> Journal of Virology. 85 (18), 9555-9567 (2011).</li><li>Van de Perre, P., 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=HIV-1+reservoirs+in+breast+milk+and+challenges+to+elimination+of+breast-feeding+transmission+of+HIV-1.">HIV-1 reservoirs in breast milk and challenges to elimination of breast-feeding transmission of HIV-1.</a> Science Translational Medicine. 4 (143), 143sr143 (2012).</li><li>Milligan, C., Richardson, B. A., John-Stewart, G., Nduati, R., Overbaugh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passively+acquired+antibody-dependent+cellular+cytotoxicity+(ADCC)+activity+in+HIV-infected+infants+is+associated+with+reduced+mortality.">Passively acquired antibody-dependent cellular cytotoxicity (ADCC) activity in HIV-infected infants is associated with reduced mortality.</a> Cell Host &amp; Microbe. 17 (4), 500-506 (2015).</li><li>Pollara, J., 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=Association+of+HIV-1+Envelope-Specific+Breast+Milk+IgA+Responses+with+Reduced+Risk+of+Postnatal+Mother-to-Child+Transmission+of+HIV-1.">Association of HIV-1 Envelope-Specific Breast Milk IgA Responses with Reduced Risk of Postnatal Mother-to-Child Transmission of HIV-1.</a> Journal of Virology. 89 (19), 9952-9961 (2015).</li><li>Ackerman, M., Nimmerjahn, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Antibody Fc. , (2014).</li><li>Huber, V. 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本文描述的用于测量ADCP活性的流式细胞学技术于2011年首次被描述30，并自此被证明为稳健型，并在80多项研究中被引用。此处描述的协议首次将该技术用于初级 BM 单元。以前对BM细胞的Fc介导功能的研究主要限于使用从对口乳分离的细胞（出生后0⁄4天）测量氧化性爆裂或基于组织学的噬菌体测定。几乎没有研究对超过隆利阶段的人类BM细胞进行过检查。使用共体细胞的研究通常?...

母乳 （BM） 由母细胞组成，这些细胞的存活率为 >90%1。细胞组成受到哺乳阶段、母婴健康状况和个人变异的强烈影响，对1、2、3、4仍然知之甚少。鉴于BM含有+103+105白细胞/mL，可以估计母乳喂养的婴儿每天摄入+105~108母性白细胞。各种体内研究表明，母性白细胞对婴儿提供关键免疫，其功能远远超过最初摄入5、6、7、8这些部位。 ，9，10，11.婴儿摄入的所有母性衍生细胞都有可能与婴儿一起发挥免疫功能或补偿婴儿自己的白细胞12。
在资源有限的国家，人体免疫缺陷病毒（艾滋病毒）的母婴传播仍然是一个危机。由于腹泻和呼吸道疾病是造成资源有限国家婴儿死亡率的显著原因，而且通过纯母乳喂养，这些疾病大大减少，这对感染艾滋病毒的母亲有利。母乳喂养远远超过风险13，14，15。除非获得清洁水和适当的婴儿配方奶粉是可靠的，否则世卫组织不建议感染艾滋病毒的母亲停止母乳喂养。每年通过 BM 进行约 100，000 次 MTCT;然而，只有15%的婴儿由其感染艾滋病毒的母亲母乳喂养感染，这表明BM17，18，19，20，21的强烈保护作用。许多因素可能协同作用，以防止传输。重要的是，BM中艾滋病毒特异性抗体（Abs）与MTCT的减少和/或减少婴儿死于艾滋病毒感染有关22，23。在很大程度上，仍不清楚的是BM细胞部分对其抗病毒特性的贡献。
许多Abs促进各种抗病毒活动，由免疫球蛋白分子的"恒定"区域，可结晶片段（Fc），通过与Fc受体（FcRs）的相互作用，发现几乎所有的先天免疫细胞，几乎所有发现在人类BM24。抗体依赖性细胞噬菌体（ADCP）已被证明是清除病毒感染所必需的，在预防艾滋病毒MTCT的情况下，已经研究不足。28,29.鉴于对BM噬菌体活性对预防艾滋病毒MTCT的潜在贡献缺乏了解，我们旨在开发一种严格的方法，将细胞从人类BM中分离，以便对由细胞介导的ADCP进行研究。BM在哺乳的不同阶段获得。

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			<td height="21" style="height:21px;width:356px;">1 &#181;m FluoSpheres NeutrAvidin-Labeled Microspheres&#160;</td>
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			<td height="21" style="height:21px;">BD Pharmingen PE Mouse Anti-Human CD45</td>
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			<td height="21" style="height:21px;">Bovine serum Albumin</td>
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			<td height="21" style="height:21px;">Corning V-bottom polystyrene 96-well plate&#160;</td>
			<td>Corning&#160;</td>
			<td style="width:85px;">3894</td>
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			<td height="21" style="height:21px;">Cytochalasin D</td>
			<td style="width:193px;">Sigma</td>
			<td>22144-77-0</td>
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			<td height="21" style="height:21px;">EZ-Link NHS-LC-LC-Biotin kit&#160;</td>
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			<td height="21" style="height:21px;width:356px;">Falcon 15mL Conical Centrifuge Tubes</td>
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			<td height="21" style="height:21px;width:356px;">Falcon 50mL Conical Centrifuge Tubes</td>
			<td style="width:193px;">Corning&#160;</td>
			<td>352070</td>
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			<td height="21" style="height:21px;">Fixable Viability Stain 450&#160;</td>
			<td style="width:193px;">BD</td>
			<td>562247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde solution</td>
			<td style="width:193px;">Sigma</td>
			<td>252549&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">HBSS without Calcium, Magnesium or Phenol Red</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:85px;">14175-095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human BD Fc Block</td>
			<td>BD</td>
			<td style="width:85px;">564219</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Serum Albumin</td>
			<td style="width:193px;">MP Biomedicals</td>
			<td style="width:85px;">2191349</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Kimtech Science Kimwipes Delicate Task Wipers</td>
			<td>Kimberly-Clark Professional&#160;</td>
			<td>34120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">PBS 1x pH 7.4</td>
			<td style="width:193px;">Thermo Fisher</td>
			<td style="width:85px;">10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Polystyrene 10mL Serological Pipettes&#160;</td>
			<td style="width:193px;">Corning&#160;</td>
			<td>4488</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein Concentrators PES, 3K MWCO, 0.5 mL</td>
			<td style="width:193px;">Pierce</td>
			<td style="width:85px;">88512</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of leukocytes from human breast milk for use in an antibody-dependent cellular phagocytosis assay of hiv targets

即使在没有抗逆转录病毒药物的情况下，只有15%的感染艾滋病毒的母亲母乳喂养的婴儿受到感染，这表明母乳（BM）具有很强的保护作用。除非获得清洁水和适当的婴儿配方奶粉是可靠的，否则世卫组织不建议感染艾滋病毒的母亲停止母乳喂养。许多因素可能协同作用，以减少 BM 传输。母乳喂养的婴儿每天摄入105~108个母性白细胞，但基本上仍不清楚这些细胞对BM抗病毒特性的贡献。 目前，我们的目标是从人类BM中分离细胞，以测量抗体依赖性细胞噬菌体（ADCP），BM噬菌体对HIV靶点最基本和普遍的先天免疫反应之一。从在哺乳不同阶段获得的5个人类BM样本中分离出细胞。分离是通过温和的离心进行，然后仔细去除乳脂和反复清洗细胞颗粒。涂有HIV包络（Env）表位的荧光珠子被用作ADCP分析的目标。细胞被染色与CD45表面标记，以识别白细胞。结果发现，ADCP活性明显高于对照实验，使用HIV特异性抗体830A可重复可测量。

牛奶可在室温或冷却器下保存（但不冷冻）;然而，鉴于我们观察到当牛奶保持非常寒冷时，其生存能力降低（数据未显示），并且在环境温度下进行短暂收集、短暂储存和运输更简单，因此建议不要冷藏样品，以减少样本到样本的可变性。在产后7-183天获得的牛奶中，由自动细胞计数器测定的细胞浓度为16，083~222，857细胞/mL。图 1说明了消除双精度?...

Watch this Scientific Journal Video about Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets at JoVE.com

Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets

母乳传播人体免疫缺陷病毒（HIV），尽管只有15%的感染艾滋病毒的母亲母乳喂养的婴儿被感染。母乳喂养的婴儿每天摄入+105+108母性白细胞，尽管这些细胞研究不足。在这里，我们描述了乳腺癌白细胞的分离，并分析了它们的噬菌体能力。

从人类母乳中分离白细胞，用于HIV靶点抗体依赖性细胞噬菌体测定

Even in the absence of antiretroviral drugs, only ~15% of infants breastfed by HIV-infected mothers become infected, suggesting a strong protective effect ...

我们开发了一种严格的方法，通过母乳噬细胞将细胞与人母乳隔离，以研究抗体依赖性细胞噬细胞病（ADCP）对HIV靶点。该技术有助于相对快速和容易分离母乳细胞，以评估白细胞群体执行ADCP的能力。演示这个程序的将是阿里萨福克斯，一个技术员从我的实验室。选择相关靶点抗原后，使用商业试剂盒根据制造商的协议对感兴趣的蛋白质进行生物素化。将生物素化样品沉积到旋转柱的上腔中。添加 PBS 至多 400 微升，盖住腔室，然后将柱插入收集管进行离心。丢弃流后，将 PBS 添加到 400 微升以进行第二次离心。丢弃流，将PBS添加到400微升，并通过分光光度计测量蛋白质浓度，将生物素蛋白与微米微球结合。在室温下，每盘联结珠用12微升的PBS中孵育6微克蛋白质，每200个PBS微升，孵育2小时，每20分钟轻轻旋转一次。在孵育结束时，通过离心将蛋白结合珠粒，然后丢弃上清液。温和涡旋后，在PBS中重新在1200微升0.1%BSA中重新补充蛋白质，并像刚刚演示的两倍通过离心洗涤管子。上次洗涤后，在 PBS 加 BSA 中将颗粒重新在 1200 微升中重新暂停。要建立ADCP检测板，首先在PBS中2%BSA中准备12微升的抗体稀释剂，或感兴趣的免疫血清，在PBS中，在一个圆底96井板中。每井向盘子中加入10微升蛋白结合珠，在37摄氏度下孵育两小时。在孵育结束时，添加200微升PBS加BSA。从使用泵表达的健康哺乳期妇女那里获得母乳后，在表达后四小时内通过离心分离出母乳含量，并小心地倒掉脱脂牛奶和脂肪，使细胞颗粒不受干扰。用无绒擦拭管内，清除管壁上的所有脂肪，并加入 10 毫升 PBS 加 HSA。用温和的移液剂重新悬浮颗粒，以避免细胞在凋亡中激活，并转移细胞悬浮液到15毫升管进行离心。在PBS加HSU中洗涤后，如刚刚证明的，轻轻地将细胞重新暂停在一到两毫升的新鲜PBS加HSU中进行计数。对于ADCP检测，在最终离心后快速反转准备好的ADCP板，将上经增验剂倒出，并在200微升PBS加BSA的第四个新鲜分离的母乳细胞中加入5倍10，在37摄氏度下孵育两小时。在孵化结束时，将细胞离心，并在200微升新鲜PBS中洗涤颗粒两次，如所证明的。上次洗涤后，在室温下，每毫升可修复的可行污渍为每毫升 0.5 微克，每井 450 微升，在室温下 30 分钟。在孵化结束时，如证明，在新鲜PBS中对细胞进行两次洗涤，并在室温下染色，使感兴趣的白细胞标记物染色30分钟，免受光线影响。在孵育结束时，收集细胞离心，并在200微升中洗两次细胞，每次洗涤1%BSA加PBS。上次洗涤后，将每井0.5%甲醛的200微升颗粒在室温下固定30分钟，防止光线。对于细胞样品的流动细胞测量分析，设置一个装有高通量板读取器的流动细胞仪，提取175微升样品，每井收集5000个细胞。执行初始浇注，以消除前进散射图与侧散点图上的双子和碎屑。使用侧面散射与生存能力染色图来消除死细胞。并使用侧散射与 CD45 图来区分主要的白细胞类。对于所有 CD45 阳性细胞，或对于感兴趣的每个白细胞子集，通过用珠阳性细胞上的标记在结合珠子的适当荧光通道的直方图中对珠阳性细胞进行浇注来测量抗体依赖性细胞吞咽活性。然后计算抗体依赖细胞吞咽率分数，作为珠阳性细胞荧光强度中位数，按总CD45加正细胞的百分比计算。此处显示了消除双精度、碎屑和死细胞的浇注策略。在总活细胞的1至12%之间通常是CD45阳性白细胞，大多数据称的侧散射低至中间CD45低单细胞似乎是前体，或不成熟细胞，基于侧散射与CD45浇注。所谓的粒细胞被定义为侧散射高，CD45中间细胞。在产后一个月内使用HIV特异性人类单克隆抗体测量新鲜分离的母乳细胞的ADCP活性表明，使用活性素抑制剂Cytochalasin D和或Fc受体在用抗体结合抗原耦合珠孵育之前使用阻断抗体的ADCP活性，导致ADCP活性在控制抗体存在时观察到或低于ADCP活性。为CD45阳性细胞确定的ADCP分数通常比背景水平高25至35倍，使用阴性对照、抗炭热单克隆抗体定义。每个母乳样本都是独一无二的，有些颗粒可能很难看到。此外，白细胞种群，因此ADCP分数，可能因样本而异。

Research

JoVE Journal

Immunology and Infection

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

科研

免疫与感染

Isolation of Brain-infiltrating Leukocytes

脑浸润白细胞的分离

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

生物能量和氧化突发：协议用于人类白细胞和血小板的隔离与评价

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

从人类母胎界面分离白细胞

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

从小鼠组织在母胎界面分离白细胞

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

从HIV-1阳性者的血浆分离外来体的

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...