Name: separation of immune cell subpopulations in peripheral blood samples from children with infectious mononucleosis
Uploaded: 2022-09-07T13:00:00
Description: Watch this Scientific Journal Video about Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis at JoVE.com

这项工作得到了国家自然科学基金（82002130）、北京自然科学基金（7222059）和中国医学科学院医学创新基金（2019-I2M-5-026）的支持。

从IM患者（n = 3），健康EBV携带者（n = 3）和未感染EBV的儿童（n = 3）获得血液样本。志愿者来自首都医科大学附属北京儿童医院，所有研究均获得伦理认可。获得首都医科大学附属北京儿童医院伦理委员会伦理认证（批准文号：[2021]-E-056-Y）。由于该研究仅使用剩余样本进行临床试验，因此放弃了患者的知情同意。所有数据都经过完全去识别化和匿名化处理，以保护患者隐私。
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<ol>
	<li>Kwok, H., Chiang, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+conventional+to+next+generation+sequencing+of+Epstein-Barr+virus+genomes.">From conventional to next generation sequencing of Epstein-Barr virus genomes.</a> Viruses. 8 (3), 60 (2016).</li><li>Bu, 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=Immunization+with+components+of+the+viral+fusion+apparatus+elicits+antibodies+that+neutralize+Epstein-Barr+virus+in+B+cells+and+epithelial+cells.">Immunization with components of the viral fusion apparatus elicits antibodies that neutralize Epstein-Barr virus in B cells and epithelial cells.</a> Immunity. 50 (5), 1305-1316 (2019).</li><li>Balfour, H. H., 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=Behavioral,+virology,+and+immunologic+factors+associated+with+acquisition+and+severity+of+primary+Epstein-Barr+virus+infection+in+university+students.">Behavioral, virology, and immunologic factors associated with acquisition and severity of primary Epstein-Barr virus infection in university students.</a> Journal of Infectious Diseases. 207 (1), 80-88 (2013).</li><li>Luzuriaga, K., Sullivan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+mononucleosis.">Infectious mononucleosis.</a> New England Journal of Medicine. 362 (21), 1993-2000 (2010).</li><li>Fujiwara, 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=Current+research+on+chronic+active+Epstein-Barr+virus+infection+in+Japan.">Current research on chronic active Epstein-Barr virus infection in Japan.</a> Pediatrics International. 56 (2), 159-166 (2014).</li><li>Tsao, S. W., Tsang, C. M., Lo, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus+infection+and+nasopharyngeal+carcinoma.">Epstein-Barr virus infection and nasopharyngeal carcinoma.</a> Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences. 372 (1732), 20160270 (2017).</li><li>Zhong, H., 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=Whole+transcriptome+profiling+reveals+major+cell+types+in+the+cellular+immune+response+against+acute+and+chronic+active+Epstein-Barr+virus+infection.">Whole transcriptome profiling reveals major cell types in the cellular immune response against acute and chronic active Epstein-Barr virus infection.</a> Scientific Reports. 7 (1), 17775 (2017).</li><li>Fedyanina, O. 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=The+nature+and+clinical+significance+of+atypical+mononuclear+cells+in+infectious+mononucleosis+caused+by+the+Epstein-Barr+virus+in+children.">The nature and clinical significance of atypical mononuclear cells in infectious mononucleosis caused by the Epstein-Barr virus in children.</a> Journal of Infectious Diseases. 223 (10), 1699-1706 (2021).</li><li>Al Tabaa, Y., 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=B-cell+polyclonal+activation+and+Epstein-Barr+viral+abortive+lytic+cycle+are+two+key+features+in+acute+infectious+mononucleosis.">B-cell polyclonal activation and Epstein-Barr viral abortive lytic cycle are two key features in acute infectious mononucleosis.</a> Journal of Clinical Virology. 52 (1), 33-37 (2011).</li><li>M, 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=Natural+Killer+cell+transcriptome+during+primary+EBV+infection+and+EBV+associated+Hodgkin+Lymphoma+in+children-A+preliminary+observation.">Natural Killer cell transcriptome during primary EBV infection and EBV associated Hodgkin Lymphoma in children-A preliminary observation.</a> Immunobiology. 225 (3), 151907 (2020).</li><li>Greenough, T. C., 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+gene+expression+signature+that+correlates+with+CD8++T+cell+expansion+in+acute+EBV+infection.">A gene expression signature that correlates with CD8+ T cell expansion in acute EBV infection.</a> Journal of Immunology. 195 (9), 4185-4197 (2015).</li><li>Xia, Y., Gao, Y., Wang, B., Zhang, H., Zhang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+the+method+of+cell+separation+from+bile+of+patients+with+cholangiocarcinoma+for+flow+cytometry.">Optimizing the method of cell separation from bile of patients with cholangiocarcinoma for flow cytometry.</a> Gastroenterology Research and Practice. , 5436961 (2019).</li><li>Maecker, H. T., Trotter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+controls,+instrument+setup,+and+the+determination+of+positivity.">Flow cytometry controls, instrument setup, and the determination of positivity.</a> Cytometry Part A. 69 (9), 1037-1042 (2006).</li><li>Byford, E., Carr, M., Pinon, L., Ahearne, M. J., Wagner, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CD4++T-cells+and+analysis+of+circulating+T-follicular+helper+(cTfh)+cell+subsets+from+peripheral+blood+using+6-color+flow+cytometry.">Isolation of CD4+ T-cells and analysis of circulating T-follicular helper (cTfh) cell subsets from peripheral blood using 6-color flow cytometry.</a> Journal of Visualized Experiments. (143), e58431 (2019).</li><li>Djaoud, Z., 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=Two+alternate+strategies+for+innate+immunity+to+Epstein-Barr+virus:+One+using+NK+cells+and+the+other+NK+cells+and+gammadelta+T+cells.">Two alternate strategies for innate immunity to Epstein-Barr virus: One using NK cells and the other NK cells and gammadelta T cells.</a> Journal of Experimental Medicine. 214 (6), 1827-1841 (2017).</li><li>Nakid-Cordero, C., Baron, M., Guihot, A., Vieillard, V. <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+in+post-transplant+lymphoproliferative+disorders.">Natural killer cells in post-transplant lymphoproliferative disorders.</a> Cancers. 13 (8), 1836 (2021).</li><li>Forrest, C., Hislop, A. D., Rickinson, A. B., Zuo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome-wide+analysis+of+CD8++T+cell+responses+to+EBV+reveals+differences+between+primary+and+persistent+infection.">Proteome-wide analysis of CD8+ T cell responses to EBV reveals differences between primary and persistent infection.</a> PLoS Pathogens. 14 (9), 1007110 (2018).</li><li>Zamai, L., 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=Understanding+the+Synergy+of+NKp46+and+co-activating+signals+in+various+NK+cell+subpopulations:+paving+the+way+for+more+successful+NK-cell-based+immunotherapy.">Understanding the Synergy of NKp46 and co-activating signals in various NK cell subpopulations: paving the way for more successful NK-cell-based immunotherapy.</a> Cells. 9 (3), 753 (2020).</li><li>Lam, J. K. 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=Emergence+of+CD4++and+CD8++polyfunctional+T+cell+responses+against+immunodominant+lytic+and+latent+EBV+antigens+in+children+with+primary+EBV+infection.">Emergence of CD4+ and CD8+ polyfunctional T cell responses against immunodominant lytic and latent EBV antigens in children with primary EBV infection.</a> Frontiers In Microbiology. 9, 416 (2018).</li><li>Choi, I. 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+the+Epstein-Barr+virus+LMP1+protein+induces+potent+cytotoxic+CD4(+)+and+CD8(+)+T+cell+responses.">Signaling by the Epstein-Barr virus LMP1 protein induces potent cytotoxic CD4(+) and CD8(+) T cell responses.</a> Proceedings of the National Academy of Sciences. 115 (4), 686-695 (2018).</li></ol>

该协议代表了对外周血免疫细胞亚群进行分类的有效方法。本研究选取IM患者、健康EBV携带者和EBV未感染儿童的静脉血样作为研究目标。本工作使用IM患者的外周血，主要侧重于通过多色流式细胞术分析和确定不同细胞亚群的比例。转录组测序主要用于检测和分析同一疾病状态下IM患儿不同免疫细胞亚群中不足以全面、特异性比较不同免疫细胞亚群的淋巴细胞亚群。因此，从外周血中分离出几种类?...

EB 病毒 （EBV） 是一种γ疱疹病毒，也称为人类疱疹病毒 4 型，在人群中普遍存在，并在 90% 以上的成年人口中建立终生潜伏感染1。大多数 EBV 原发性感染发生在儿童期和青春期，少数患者表现为传染性单核细胞增多症 （IM）2，表现出特征性免疫病理学，包括血液中 CD8+ T 细胞的激活免疫应答和口咽部 EBV 感染的 B 细胞短暂增殖3。IM的病程可能持续2-6周，大多数患者恢复良好4。然而，有些人会出现持续性或复发性IM样症状，发病率和死亡率很高，这被归类为慢性活动性EBV感染（CAEBV）5。此外，EB病毒是一种重要的致癌病毒，与多种恶性肿瘤密切相关，包括鼻咽癌、伯基特淋巴瘤、霍奇金淋巴瘤（HL）和T/NK细胞淋巴瘤6等上皮样和淋巴恶性肿瘤。尽管EB病毒已经研究了50多年，但其发病机制和诱导淋巴细胞增殖的机制尚未完全阐明。
一些研究已经通过转录组测序研究了EBV感染免疫病理学的分子特征。钟等分析了中国IM或CAEBV患儿外周血单核细胞（PBMC）的全转录组分析，发现CD8+T细胞扩增主要见于IM组7，提示CD8+T细胞可能在IM中起主要作用。同样，另一项研究发现，与由EBV再激活和其他药物引起的IM患者相比，原发性EBV感染引起的IM患者中EBV特异性细胞毒性T和CD19 + B细胞的比例较低，CD8 + T细胞的百分比较高8。B细胞首先参与IM，因为EBV受体存在于其表面。Al Tabaa等人发现B细胞在IM9期间被多克隆激活和分化的原浆母细胞（CD19+，CD27+和CD20−以及CD138−细胞）和浆细胞（CD19+，CD27+和CD20−以及CD138+）。此外，Zhong等人发现单核细胞标志物CD14和CD64在CAEBV中上调，表明单核细胞可能通过抗体依赖性细胞毒性（ADCC）和过度活跃的吞噬作用在CAEBV的细胞免疫应答中发挥重要作用7。Alka等人对来自四名IM或HL患者的MACS分选CD56暗CD16 + NK细胞的转录组进行了表征，发现来自IM和HL的NK细胞具有下调的先天免疫和趋化因子信号基因，这可能是NK细胞低反应的原因10。此外，Greenough等人分析了来自IM个体的10个PBMC的分选CD8 + T细胞的基因表达。他们报告说，IM中很大一部分CD8 + T细胞具有病毒特异性，激活，分裂，并已准备好发挥效应活性11。在原发性 EBV 感染期间，T 细胞介导的 EBV 特异性反应和 NK 细胞介导的非特异性应答都起着至关重要的作用。然而，这些研究仅研究了免疫细胞多种混合物或仅研究特定淋巴细胞亚群的转录组结果，不足以全面比较同一疾病状态下IM患儿不同免疫细胞亚群的分子特征和功能。
本文描述了一种结合免疫磁珠和荧光激活细胞分选（FACS）来分离和分析PBMC的明确免疫细胞亚群（单核细胞，CD4 + T细胞，CD8 + T细胞，B细胞和NK细胞）的方法。使用这种方法，可以使用磁选机和FACS纯化磁性和荧光标记的细胞或通过流式细胞术进行分析。可以从纯化的细胞中提取RNA进行转录组测序。该方法将使IM个体在相同疾病状态下的不同免疫细胞的表征和基因表达成为可能，这将扩展我们对EBV感染免疫病理学的理解。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>APC anti-human CD16</td>
			<td>Biolegend</td>
			<td>302012</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>APC anti-human CD56 (NCAM)</td>
			<td>Biolegend</td>
			<td>362504</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>APC/Cyanine7 anti-human CD4</td>
			<td>Biolegend</td>
			<td>344616</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>Automated cell counter</td>
			<td>BIO RAD</td>
			<td>TC20</td>
			<td>Cell count</td>
		</tr>
		<tr>
			<td>BD FACSAria fluorescence-activated&#160;flow&#160;cell&#160;sorter-cytometer (BD FACSAria II)</td>
			<td>Becton, Dickinson and Company</td>
			<td>644832</td>
			<td>Cell sort</td>
		</tr>
		<tr>
			<td>CD14 MicroBeads, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-050-201</td>
			<td>microbeads</td>
		</tr>
		<tr>
			<td>Cell ctng slides</td>
			<td>BIO RAD</td>
			<td>1450016</td>
			<td>Cell count</td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>Falcon</td>
			<td>35209715</td>
			<td>15 mL centrifuge tube</td>
		</tr>
		<tr>
			<td>EDTA (&#8805;99%, BioPremium)</td>
			<td>Beyotime</td>
			<td>ST1303</td>
			<td>EDTA</td>
		</tr>
		<tr>
			<td>Ethylene diamine tetra acetic acid (EDTA) anticoagulant tubes</td>
			<td>Becton, Dickinson and Company</td>
			<td>&#160;367862&#160;</td>
			<td>EDTA anticoagulant tubes</td>
		</tr>
		<tr>
			<td>FITC anti-human CD19</td>
			<td>Biolegend</td>
			<td>302206</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>Gibco Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000-044</td>
			<td>Fetal Bovine Serum</td>
		</tr>
		<tr>
			<td>&#160;High-speed&#160;centrifuge</td>
			<td>Sigma</td>
			<td>&#160;3K15</td>
			<td>Cell centrifugation for 15 mL centrifuge tube</td>
		</tr>
		<tr>
			<td>&#160;High-speed&#160;centrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td>Cell centrifugation for 1.5 mL Eppendorf (EP) tube</td>
		</tr>
		<tr>
			<td>Human lymphocyte separation medium</td>
			<td>Dakewe</td>
			<td>DKW-KLSH-0100</td>
			<td>Ficoll-Paque</td>
		</tr>
		<tr>
			<td>LS&#160;Separation&#160;columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>Separation&#160;columns</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td>1.5 mL microcentrifuge tube</td>
		</tr>
		<tr>
			<td>MidiMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td>Magnetic bead separator</td>
		</tr>
		<tr>
			<td>PE anti-human CD8</td>
			<td>Biolegend</td>
			<td>344706</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>PerCP/Cyanine5.5 anti-human CD3</td>
			<td>Biolegend</td>
			<td>344808</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>BI</td>
			<td>02-024-1ACS</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Polystyrene round bottom tubes</td>
			<td>Falcon</td>
			<td>352235</td>
			<td>5 mL tube for FACS flow cytometer</td>
		</tr>
		<tr>
			<td>TRIzol reagent</td>
			<td>Ambion</td>
			<td>15596024</td>
			<td>Lyse cells for RNA extraction</td>
		</tr>
		<tr>
			<td>Trypan Blue Staining Cell Viability Assay Kit</td>
			<td>Beyotime</td>
			<td>C0011</td>
			<td>Trypan Blue Staining</td>
		</tr>
	</tbody>
</table>

separation of immune cell subpopulations in peripheral blood samples from children with infectious mononucleosis

传染性单核细胞增多症 （IM） 是一种急性综合征，主要与原发性 EB 病毒 （EBV） 感染有关。主要临床症状包括不规则发热、淋巴结肿大和外周血淋巴细胞明显增多。IM的致病机制尚不清楚;目前尚无有效的治疗方法，主要有对症治疗。EBV免疫生物学中的主要问题是，为什么只有一小部分感染者表现出严重的临床症状，甚至发展为EBV相关的恶性肿瘤，而大多数人在病毒中终生无症状。
B细胞首先参与IM，因为EBV受体存在于其表面。自然杀伤（NK）细胞是细胞毒性先天淋巴细胞，对杀死EBV感染的细胞很重要。在急性EBV感染期间，CD4 + T细胞的比例降低，而CD8 + T细胞的比例急剧扩大，CD8 + T细胞的持久性对于IM的终身控制很重要。这些免疫细胞在IM中起着重要作用，它们的功能需要单独识别。为此，首先使用CD14微珠，色谱柱和磁分离器从IM个体的外周血单核细胞（PBMC）中分离单核细胞。
其余PBMC用peridinin-叶绿素蛋白（PerCP）/花青素5.5抗CD3、别藻蓝蛋白（APC）/花青7抗CD4、藻红蛋白（PE）抗CD8、异硫氰酸荧光素（FITC）抗CD19、APC抗CD56和APC抗CD16抗体染色，以使用流式细胞仪分选CD4 + T细胞，CD8 + T细胞，B细胞和NK细胞。此外，对5个亚群进行转录组测序，探讨其在IM中的功能和致病机制。

门控策略参考 用于对四个淋巴细胞亚群进行分类的门控策略如图1所示。简而言之，在显示颗粒度（SSC-A）与大小（FSC-A）的点图上选择淋巴细胞（P1）。然后，在显示大小（FSC-A）与前向散射（FSC-H）的点图上选择单个单元格（P2），同时排除双联单元格。CD3 + T细胞（P3）和CD19 + B细胞（图1B）在点图上分别选择，显示CD3 Per...

Watch this Scientific Journal Video about Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis at JoVE.com

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

我们描述了一种结合免疫磁珠和荧光激活细胞分选的方法，以分离和分析外周血单核细胞（单核细胞，CD4 + T细胞，CD8 + T细胞，B细胞和自然杀伤细胞）的已定义免疫细胞亚群。使用这种方法，可以纯化和分析磁性和荧光标记的细胞。

传染性单核细胞增多症儿童外周血样本中免疫细胞亚群的分离

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

该方法将能够表征IM个体处于相同疾病状态的不同免疫细胞，这将扩展我们对EBV感染机制的理解。演示该程序的将是来自我们实验室的医生张琳琳。首先，将先前分离的PBMC置于1.5毫升微量离心管中，并在室温下以300G旋转10分钟。弃去上清液，并用80微升制备的缓冲液重新悬浮细胞。接下来，向细胞悬液中加入20微升CD 14微珠，通过移液充分混合，并将管在冰上孵育15分钟。然后使用一毫升缓冲液洗涤PBMC，并在室温下以300G离心10分钟。弃去整个上清液，并用500微升缓冲液重新悬浮细胞。对于磁分离，将一根色谱柱放在磁珠分离器上，然后用三毫升缓冲液洗涤色谱柱。接下来，将细胞悬液添加到列中。让它通过，并将未标记的细胞收集到15毫升离心管中。用三毫升缓冲液洗涤柱三次，并将整个流出物收集在15毫升离心管中。现在，将色谱柱放入新的15毫升离心管中，并向色谱柱中加入5毫升缓冲液。将柱塞牢固地推入色谱柱中，将磁性标记的细胞冲洗到管中。将磁性标记的细胞以300 G离心五分钟。除去上清液后，将细胞重新悬浮在500微升PBS中，并将细胞悬液转移到1.5毫升微量离心管中进行后续转录组测序。通过以300G离心5分钟来沉淀收集的未标记细胞，并将细胞重新悬浮在1.5毫升微量离心管中的100微升PBS中。然后，向细胞悬液中加入两微升每种标记的抗体，并在冰上孵育30分钟，避光。接下来，将100微升PBMC细胞悬液等分到1.5毫升微量离心管中，以设置阴性对照样品CD 3，CD 4，CD 8和CD 19，单染色样品和CD 56或CD 16染色样品。然后向每个试管中加入两微升相应的荧光标记抗体，并在冰上孵育30分钟，避光。孵育后，如前所述用PBS洗涤所有细胞，并重新悬浮在500微升PBS中。打开细胞分选系统，然后运行开机程序。然后，安装85微米喷嘴，并打开分拣流。将分拣电压设置为 4， 500 伏，将频率设置为 47。在断路窗中将间隙设置为8，开启最佳点自动分选模式，自动确定液滴振幅值。最后，在侧流窗口中将液滴延迟调整为 30.31。使用前向散射点图与侧向散射点图，绘制多边形门 P1 以识别完整的淋巴细胞群。接下来，使用前向散点面积与高度点图，绘制多边形门 P2 以标识单个像元，并排除双峰。然后使用CD 19 FITC面积与CD 3每个CPCY 5.5面积点图，绘制矩形门P3以选择CD 3阳性T细胞和CD 19阳性B细胞。接下来，使用CD 4 APCCY 7区域与CD 8 PE区域点图，绘制矩形门以选择CD 4和CD 8阳性T细胞。最后，使用侧散射区域与CD 56或CD 16 APC面积点图，绘制矩形门以选择CD 56或CD 16阳性自然杀伤细胞。对于阴性对照管，单击采集仪表板中的加载，然后在软件中选择细胞仪设置。在检查器窗口中，单击参数选项卡，然后调整正向散射、侧向散射和不同荧光染料的电压。将单染色管装入细胞仪后，单击采集仪表板中的加载，然后单击补偿选项卡以调整补偿，如文本中所述。轻轻涡旋细胞悬液，然后将试管装入细胞仪。将200微升FBS加入四个收集流管中，并涡旋管。将它们放入细胞仪收集室中。在四个流管中分别收集不同类型的细胞。将分离的细胞以 300 G 离心五分钟，然后将 200 μL RNA 分离试剂添加到细胞调色板中以进行转录组测序。本研究采用免疫磁珠和荧光活化细胞分选技术，从不同样本的外周血中高效分选免疫细胞亚群。重要的是，在该协议中，细胞分选步骤已经过优化以提高细胞活力。与健康的EB病毒携带者和未感染的样本相比，分选的免疫细胞显示传染性单核细胞增多症患者中CD3，CD 8阳性T细胞的比例增加。相比之下，与健康EBV携带者和未感染EBV的儿童相比，在传染性单核细胞增多症患者中观察到CD 3，CD 4阳性T细胞和CD 19阳性B细胞的比例降低。此外，与未感染EBV的儿童相比，在传染性单核细胞增多症患者中观察到CD 56或CD 16阳性自然杀伤细胞的比例降低。保持细胞活力对于后续实验至关重要。在分选过程中将细胞保存在冰上或冰箱中有利于细胞活力。

Research

JoVE Journal

Immunology and Infection

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

枚举主要外周血白细胞人口多中心临床试验，使用全血表型分析

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

科研

免疫与感染

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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 ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...