Name: measuring naturally acquired phagocytosis-inducing antibodies to plasmodium falciparum parasites by a flow cytometry-based assay
Uploaded: 2020-08-06T16:20:00
Description: Watch this Scientific Journal Video about Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay at JoVE.com

迈肯·维斯蒂感谢出色的技术援助。这项工作部分由丹麦外交部的赠款（MAVARECA II;17-02-KU）供资，由丹妮达研究金中心管理。资助者在研究设计、数据收集和分析、决定出版或准备手稿方面没有作用。

用于此处结果的人体血清样本在另外一项研究19中收集。馆藏由加纳大学野口医学研究所机构审查委员会（研究报告038/10-11）和丹麦首都大区区域研究伦理委员会（协议H-4-2013-083）批准。
1. 寄生虫文化
注：遵守当地有关人类病原体处理的规定。
<ol><li>根据寄生虫培养基20前所述的标准协议维持恶性疟原虫（参见20</su...

<ol>
	<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=World+Malaria+Report+-+2018.">World Malaria Report - 2018.</a> World Health Organization. , (2018).</li><li>Pierce, S. K., Miller, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=World+Malaria+Day+2009:+What+malaria+knows+about+the+immune+system+that+immunologists+still+do+not.">World Malaria Day 2009: What malaria knows about the immune system that immunologists still do not.</a> Journal of Immunology. 182 (9), 5171-5177 (2009).</li><li>Gamain, B., Smith, J. D., Viebig, N. K., Gysin, J., Scherf, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pregnancy-associated+malaria:+Parasite+binding,+natural+immunity+and+vaccine+development.">Pregnancy-associated malaria: Parasite binding, natural immunity and vaccine development.</a> International Journal for Parasitology. 37 (3-4), 273-283 (2007).</li><li>Staalsoe, 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=Variant+surface+antigen-specific+IgG+and+protection+against+clinical+consequences+of+pregnancy-associated+Plasmodium+falciparum+malaria.">Variant surface antigen-specific IgG and protection against clinical consequences of pregnancy-associated Plasmodium falciparum malaria.</a> Lancet. 363 (9405), 283-289 (2004).</li><li>Feng, 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=Antibodies+to+variant+surface+antigens+of+plasmodium+falciparum+-Infected+erythrocytes+are+associated+with+protection+from+treatment+failure+and+the+development+of+anemia+in+pregnancy.">Antibodies to variant surface antigens of plasmodium falciparum -Infected erythrocytes are associated with protection from treatment failure and the development of anemia in pregnancy.</a> The Journal of Infectious Diseases. 200 (2), 299-306 (2009).</li><li>Teo, A., Feng, G., Brown, G. V., Beeson, J. G., Rogerson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Antibodies+and+Protection+against+Blood-stage+Malaria.">Functional Antibodies and Protection against Blood-stage Malaria.</a> Trends in Parasitology. 32 (11), 1-12 (2016).</li><li>Aitken, E. H., Mahanty, S., Rogerson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+effector+functions+in+malaria+and+other+parasitic+diseases:+a+few+needles+and+many+haystacks.">Antibody effector functions in malaria and other parasitic diseases: a few needles and many haystacks.</a> Immunology &amp; Cell Biology. 98 (4), 264-275 (2020).</li><li>Celada, A., Cruchaud, A., Perrin, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opsonic+activity+of+human+immune+serum+on+in+vitro+phagocytosis+of+Plasmodium+falciparum+infected+red+blood+cells+by+monocytes.">Opsonic activity of human immune serum on in vitro phagocytosis of Plasmodium falciparum infected red blood cells by monocytes.</a> Clinical and Experimental Immunology. 47 (3), 635-644 (1982).</li><li>Celada, A., Cruchaud, A., Perrin, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+of+Plasmodium+falciparum-Parasitized+Erythrocytes+by+Human+Polymorphonuclear+Leukocytes.">Phagocytosis of Plasmodium falciparum-Parasitized Erythrocytes by Human Polymorphonuclear Leukocytes.</a> The Journal of Parasitology. 69 (1), 49-53 (1983).</li><li>Jaworowski, 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=Relationship+between+human+immunodeficiency+virus+type+1+coinfection,+anemia,+and+levels+and+function+of+antibodies+to+variant+surface+antigens+in+pregnancy-associated+malaria.">Relationship between human immunodeficiency virus type 1 coinfection, anemia, and levels and function of antibodies to variant surface antigens in pregnancy-associated malaria.</a> Clinical and Vaccine Immunology. 16 (3), 312-319 (2009).</li><li>Ata&#237;de, R., Mwapasa, V., Molyneux, M. E., Meshnick, S. R., Rogerson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+that+induce+phagocytosis+of+malaria+infected+erythrocytes:+Effect+of+HIV+infection+and+correlation+with+clinical+outcomes.">Antibodies that induce phagocytosis of malaria infected erythrocytes: Effect of HIV infection and correlation with clinical outcomes.</a> PLoS One. 6 (7), 22491 (2011).</li><li>Ghumra, 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=Immunisation+with+recombinant+PfEMP1+domains+elicits+functional+rosette-inhibiting+and+phagocytosis-inducing+antibodies+to+Plasmodium+falciparum.">Immunisation with recombinant PfEMP1 domains elicits functional rosette-inhibiting and phagocytosis-inducing antibodies to Plasmodium falciparum.</a> PloS One. 6 (1), 0016414 (2011).</li><li>Ghumra, 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=Induction+of+strain-transcending+antibodies+against+Group+A+PfEMP1+surface+antigens+from+virulent+malaria+parasites.">Induction of strain-transcending antibodies against Group A PfEMP1 surface antigens from virulent malaria parasites.</a> PLoS Pathogens. 8 (4), 1002665 (2012).</li><li>Chan, J. 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=Targets+of+antibodies+against+erythrocytes+in+malaria+immunity.">Targets of antibodies against erythrocytes in malaria immunity.</a> J Clin Invest. 122 (9), 3227-3238 (2012).</li><li>Quintana, M. P., Angeletti, D., Moll, K., Chen, Q., Wahlgren, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+inducing+antibodies+to+Plasmodium+falciparum+upon+immunization+with+a+recombinant+PfEMP1+NTS+DBL1&#945;+domain.">Phagocytosis inducing antibodies to Plasmodium falciparum upon immunization with a recombinant PfEMP1 NTS DBL1&#945; domain.</a> Malaria Journal. 1, 1-9 (2016).</li><li>Chan, J. 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=A+single+point+in+protein+trafficking+by+Plasmodium+falciparum+determines+the+expression+of+major+antigens+on+the+surface+of+infected+erythrocytes+targeted+by+human+antibodies.">A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.</a> Cellular and Molecular Life Sciences. 73, 4141-4158 (2016).</li><li>Quintana, M. 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=Antibodies+in+children+with+malaria+to+PfEMP1,+RIFIN+and+SURFIN+expressed+at+the+Plasmodium+falciparum+parasitized+red+blood+cell+surface.">Antibodies in children with malaria to PfEMP1, RIFIN and SURFIN expressed at the Plasmodium falciparum parasitized red blood cell surface.</a> Scientific Reports. 8 (1), 3262 (2018).</li><li>Hommel, 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=Evaluating+antibody+functional+activity+and+strain-specificity+of+vaccine+candidates+for+malaria+in+pregnancy+using+in+vitro+phagocytosis+assays.">Evaluating antibody functional activity and strain-specificity of vaccine candidates for malaria in pregnancy using in vitro phagocytosis assays.</a> Parasites &amp; Vectors. 11 (69), 1-7 (2018).</li><li>Ampomah, P., Stevenson, L., Ofori, M. F., Barfod, L., Hviid, L. <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+responses+to+pregnancy-restricted+and+-unrestricted+Plasmodium+falciparum+erythrocyte+membrane+protein+1+antigens+in+Ghanaian+women+naturally+exposed+to+malaria+parasites.">B-cell responses to pregnancy-restricted and -unrestricted Plasmodium falciparum erythrocyte membrane protein 1 antigens in Ghanaian women naturally exposed to malaria parasites.</a> Infection and Immunity. 82 (5), 1860-1871 (2014).</li><li>Cranmer, S. L., Magowan, C., Liang, J., Coppel, R. L., Cooke, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+to+serum+for+cultivation+of+Plasmodium+falciparum+in+vitro.">An alternative to serum for cultivation of Plasmodium falciparum in vitro.</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 91 (3), 363-365 (1997).</li><li>Lambros, C., Vanderberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+Plasmodium+falciparum+erythrocytic+stages+in+culture.">Synchronization of Plasmodium falciparum erythrocytic stages in culture.</a> Journal of Parasitology. 65 (3), 418-420 (1979).</li><li>Barfod, 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=Human+pregnancy-associated+malaria-specific+B+cells+target+polymorphic,+conformational+epitopes+in+VAR2CSA.">Human pregnancy-associated malaria-specific B cells target polymorphic, conformational epitopes in VAR2CSA.</a> Molecular Microbiology. 63 (2), 335-347 (2007).</li><li>Staalsoe, 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=In+vitro+selection+of+Plasmodium+falciparum+3D7+for+expression+of+variant+surface+antigens+associated+with+severe+malaria+in+African+children.">In vitro selection of Plasmodium falciparum 3D7 for expression of variant surface antigens associated with severe malaria in African children.</a> Parasite Immunology. 25 (8-9), 421-427 (2003).</li><li>Chan, 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=Regulation+of+PfEMP1-VAR2CSA+translation+by+a+Plasmodium+translation-enhancing+factor.">Regulation of PfEMP1-VAR2CSA translation by a Plasmodium translation-enhancing factor.</a> Nature Microbiology. 2, 17068 (2017).</li><li>Barfod, 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=Evasion+of+immunity+to+Plasmodium+falciparum+malaria+by+IgM+masking+of+protective+IgG+epitopes+in+infected+erythrocyte+surface-exposed+PfEMP1.">Evasion of immunity to Plasmodium falciparum malaria by IgM masking of protective IgG epitopes in infected erythrocyte surface-exposed PfEMP1.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (30), 12485-12490 (2011).</li><li>Moll, K., Kaneko, A., Scherf, A., Wahlgren, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Malaria Research. , (2013).</li><li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+malaria+microscopy.+Part+I.+Learner's+Guide.">Basic malaria microscopy. Part I. Learner's Guide.</a> World Health Organization. , (1991).</li><li>Tsuchiya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+human+acute+monocytic+leukemia+cell+line+(THP-1).">Establishment and characterization of a human acute monocytic leukemia cell line (THP-1).</a> International Journal of Cancer. Journal International Du Cancer. 26 (2), 171-176 (1980).</li><li>Fleit, H. B., Kobasiuk, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+monocyte-like+cell+line+THP-1+expresses+FcyRl+and+Fc'yRIl.">The human monocyte-like cell line THP-1 expresses FcyRl and Fc'yRIl.</a> Journal of Leukocyte Biology. 49, 556-565 (1991).</li><li>Auwerx, J., Staels, B., Van Vaeck, F., Ceuppens, 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=Changes+in+IgG+Fc+receptor+expression+induced+by+phorbol+12-myristate+13-acetate+treatment+of+THP-1+monocytic+leukemia+cells.">Changes in IgG Fc receptor expression induced by phorbol 12-myristate 13-acetate treatment of THP-1 monocytic leukemia cells.</a> Leukemia research. 16 (3), 317-327 (1992).</li><li>Ata&#237;de, 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=Using+an+improved+phagocytosis+assay+to+evaluate+the+effect+of+HIV+on+specific+antibodies+to+pregnancy-associated+malaria.">Using an improved phagocytosis assay to evaluate the effect of HIV on specific antibodies to pregnancy-associated malaria.</a> PloS One. 5 (5), 10807 (2010).</li></ol>

这里提出的协议先前已经描述，并使用,12，15，17，31,31测量抗体的能力，针对P.恶性疟原虫的表层，以诱导蛋白位和吞噬的THP-1细胞。15,此处提供的结果侧重于生活在恶性疟原虫流行地区的妇女的血浆/血清中自然获得的VAR2CSA特异性抗体。VAR2CSA 是一种参与 IEs 的塞盘固存的 PfEMP1 类型，是 ...

疟疾是一种病媒传播的疾病，在感染五种不同种类的疟原虫时，由人类引起。最常见的物种是恶性疟原虫，它也负责发病率和死亡率1。疟疾临床表现从无症状或良性感染到复杂/严重疾病，后者主要发生在五岁以下儿童中。接触恶性疟原虫不会诱发无菌免疫，但生活在流行地区的个体对临床疾病的免疫力会慢慢发展。保护是年龄/暴露依赖和免疫力通常获得在生命的前5-10年2。成年妇女是一个重要的例外，因为严重的疟疾可能发生在怀孕期间，在临床介绍称为胎儿素疟疾（PM）。PM 是流产、死产、早产、低出生体重、胎儿死亡和产妇贫血的重要原因。对PM的耐药性在连续怀孕时发展 3。防止PM与获得抗体对VAR2CSA型PfEMP14，5，受感染的红细胞（IE）表面抗原，结合到软体素硫酸盐A（CSA），使国际在胎盘中固有。4,抗体调解保护执行各种功能活动（审查在,6，7），包括E的异化，以诱导吞噬。早期的体外研究表明，抗体可以通过,噬菌体8，9来限制单细胞的存在。9最近的研究表明，高浓度的噬菌体诱导抗体与更好的妊娠结果（在HIV共感染的情况下）10，11，,11表明这种效应功能在自然获得的免疫反应的相关性。
在这里，我们提出了一个协议，测量存在于人类血浆/血清中的抗体的这一功能in vitro，使用体外培养的IES表达VAR2CSA与单细胞线THP-1。与早期的显微镜方案8相比,,，该测定方法以前曾使用过,,,11、12、13、14、15、16、17、18，被认为是一种改进12和更容易的方法，因为它允许使用较小的抗体体积在一次运行中测试更多的抗体样本，并避免繁琐和偏颇的18显微镜计数。17,1113141516尽管该测定方法已被多个实验室使用，而且执行过程非常简单，但它需要仔细规划和准备，因此，详细的协议将允许缺乏经验的实验室和研究人员应用该测定。例如，我们使用表示 VAR2CSA 的后期同步 IE 与从自然获得 PM 特异性免疫力的妇女收集的血清中存在的抗体进行结果化。然而，该协议可以很容易地修改，以检测抗体的功能，任何寄生虫抗原存在于国际化学品表面，无论是由自然接触或接种疫苗引起的。

<table>
	<tbody>
		<tr>
			<td>96 well cell culture plates, round bottom with lid</td>
			<td>Corning</td>
			<td>3799</td>
			<td>Any similar plate can be used, make sure it is compatible with the flow cytometer instrument you intend to use</td>
		</tr>
		<tr>
			<td>AlbuMAX-II</td>
			<td>Gibco</td>
			<td>11021-037</td>
			<td></td>
		</tr>
		<tr>
			<td>AlbuMAX-II (5%)</td>
			<td>-</td>
			<td>-</td>
			<td>5% AlbuMAX-II (Gibco, 11021-037), 0.2g/L hypoxanthine (Sigma, H9377) in RPMI1640 (Sigma, R5886)</td>
		</tr>
		<tr>
			<td>Anti-Red Blood Cells antibody</td>
			<td>Abcam</td>
			<td>ab34858</td>
			<td>Prepare 2&#65533;&#65533;l aliquots and freeze a -20&#176;C. Use one aliquot per experiment.</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Sigma</td>
			<td>P8622</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Protein G</td>
			<td>Invitrogen</td>
			<td>10003D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide solution</td>
			<td>Sigma</td>
			<td>E1510</td>
			<td>Prepare a stock solution at 0.1mg/mL in RPMI1640 (R5886). Store protected from light</td>
		</tr>
		<tr>
			<td>FC500 flow cytometer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Any flow cytometer supporting 96 well plate format and having the appropriate lasers/filters to measure EtBr fluorescence can be used.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10099-141</td>
			<td>Heat inactivate before use.</td>
		</tr>
		<tr>
			<td>FITC mouse anti-human CD16</td>
			<td>BD Biosciences</td>
			<td>555406 or 556618</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC mouse anti-human CD32</td>
			<td>BD Biosciences</td>
			<td>552883</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC mouse anti-human CD64</td>
			<td>BD Biosciences</td>
			<td>555527</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowLogic software</td>
			<td>Inivai technologies</td>
			<td></td>
			<td>Any flow cytometry analysis can be used, for example FlowJo or Winlist</td>
		</tr>
		<tr>
			<td>Gentamicin (10mg/mL)</td>
			<td>Sigma</td>
			<td>G1272</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxanthine</td>
			<td>Sigma</td>
			<td>H9377</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine (200mM)</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing solution</td>
			<td>-</td>
			<td>-</td>
			<td>15mM NH4Cl, 10mM NaHCO3, 1mM EDTA</td>
		</tr>
		<tr>
			<td>MACS CS-column and accesories</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-305</td>
			<td></td>
		</tr>
		<tr>
			<td>Parasite culture medium</td>
			<td>-</td>
			<td>-</td>
			<td>2mM L-glutamine (Sigma, G7513), 50&#181;g/mL Gentamicin (Sigma, G1272), 0.5% AlbuMAX-II (AlbuMAX-II 5%) in RPMI1640 (Sigma, R5886)</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (10000U and 10mg/mL)</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>Sigma</td>
			<td>R5886</td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1 culture medium</td>
			<td>-</td>
			<td>-</td>
			<td>10%FBS (Gibco, 10099-141), 2mM L-glutamine (Sigma, G7513), 100U/mL Penicillin, 0.1mg/mL Streptomycin (Sigma, P0781) in RPMI1640 (Sigma, R5886)</td>
		</tr>
		<tr>
			<td>Vario MACS magnet</td>
			<td>Miltenyi Biotec</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring naturally acquired phagocytosis-inducing antibodies to plasmodium falciparum parasites by a flow cytometry-based assay

该协议描述了如何建立和运行一个流细胞学为基础的噬菌体噬菌体测定恶性疟原虫 - 感染红细胞 （IE） 由自然获得的IgG抗体特异性VAR2CSA。VAR2CSA是一种寄生虫抗原，它调解胎盘中可引起严重疟疾的胎盘内 IEs 的选择性固原，称为胎盘疟疾 （PM）。防止PM由VAR2CSA特异性抗体进行调解，这些抗体被认为通过抑制地盘固存和/或通过治疗吞咽病的性病来发挥作用。该检测采用体外选择的晚期同步性，以表达VAR2CSA、in vitro具有自然获得PM特异性免疫力的妇女的血浆/血清抗体，以及噬菌体细胞系THP-1。然而，该协议可以很容易地修改，以检测抗体的功能，任何寄生虫抗原存在于国际化学品表面，无论是由自然接触或接种疫苗引起的。该测定方法对疟疾抗体中量免疫的一个重要功能方面进行了简单、高通量的评价，具有良好的可重复性。因此，在评估恶性疟原虫疟疾的临床免疫力时，它很有用，恶性疟原虫是热带地区，特别是撒哈拉以南非洲地区发病率和死亡率的主要原因。

在这里，我们详细介绍了一个协议，以前描述31，并使用11，12，13，14，15，16，17，18,14,15,16,17,1811,12,测量抗体的能力，目标为P.恶性疟原虫的表，诱导蛋白石化和吞噬的THP...

Watch this Scientific Journal Video about Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay at JoVE.com

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

该协议的总体目标是指导如何测量自然接触恶性疟原虫感染的血清或血浆中的抗体的能力，以对抗和诱导寄生虫感染的红细胞（IEs）的噬菌体。 Plasmodium falciparum

通过流细胞学测定，测量自然获得的噬菌体诱导恶性疟Plasmodium falciparum原虫抗体

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

该协议允许测量抗体对恶性疟原虫感染红细胞的吞咽和诱导细胞病的能力。可测试自然接触寄生虫感染的个体血清中的抗体，以及由寄生虫抗原免疫引起的抗体。与我一起演示这个程序的将是我实验室的技术人员迈肯·维斯蒂。为了建立一个THP-1细胞培养物进行噬菌体测定，在实验前一天将细胞播种在25厘米方培养瓶中，浓度为2.5倍，10至5.5细胞，在THP一个细胞培养基中。在实验当天，在实验开始前至少一小时，每孔PBS中用150微升无菌2%FBS的圆底96孔板进行圆底。对于中晚期受三磷酸盐感染的红细胞的收获和纯化，在10毫升寄生虫培养基中准备约1.2毫升的包装P恶性疟原虫培养物，至少含有5%的寄生虫，并加入磁柱。用50毫升的寄生虫培养培养培养剂清洗柱。要清除受感染的红细胞，请从磁铁上取下柱，用50毫升的寄生虫培养培养介质冲洗。然后，通过离心收集受感染的红细胞，将颗粒重新塞在一毫升新鲜寄生虫培养介质中进行计数。将纯化受感染的红细胞悬浮液以含有溴化乙基的寄生虫培养基中每毫升第七细胞的3.3倍10调整为每毫升2.5微克的最终浓度。接下来，将板轻拂到废容器中，从一个 96 井板中去除阻塞溶液。然后去除纸巾上多余的，并添加30微升的溴化乙基标签感染红细胞悬浮液到所有，但一个井在板的上半部分。在空井中加入30微升寄生虫培养培养剂，在室温下孵育板10分钟，防止光线。在孵育结束时，在每井中加入170微升寄生虫培养培养，通过离心沉淀被感染的红细胞。将上流水原轻拂到废物容器中，再清洗两次溴化乙基标记的受感染红细胞，每井有200微升的寄生虫培养培养基，刚才证明了这一点。第二次洗涤后，使用多通道移液器小心地从每一个井中去除全量的上流液，而不会干扰颗粒。并在30微升抗体溶液中重新暂停溴化乙酰胺标记的感染红细胞，包括以前在寄生虫培养基中准备的适当对照和测试样品。然后，将板在37摄氏度下放置45分钟，防止光线。当受感染的红细胞被解化时，通过离心收集THP-1细胞，并在12毫升新鲜、预热的THP-1细胞培养物中重新供应颗粒。第二次离心后，将颗粒重新在THP-1细胞培养的一毫升中重新加量进行计数，并在THP-1细胞培养中将细胞浓度调整为每毫升5倍至第5个细胞。从第二个96井板中去除阻解溶液，并将100微升的THP-1电池添加到每个井中。然后，将板放在细胞培养箱中。在蛋白化孵化结束时，在蛋白化板的每个井中加入170微升的寄生虫培养培养，通过离心将受感染的红细胞沉淀到板块底部。每井用200微升的寄生虫培养剂两次清洗被感染的红细胞，使用多通道移液器在第二次洗涤后小心地去除上经剂。在每井预加热的THP-1细胞培养物的100微升中重新悬浮受作用的受感染红细胞，并每井将50微升的受作用的受感染红细胞悬浮液转移到吞噬细胞细胞的对应井中。添加所有细胞后，将板放在细胞培养箱中不超过 40 分钟，防止光线。在孵育结束时，在摄氏四度时离心，停止吞噬。去除上一甲酸，每井用150微升的室温氯化铵解溶液重新预支颗粒。整整三分钟后，在PBS中加入100微升的冰冷2%FBS，以阻止解变，通过离心沉淀完好的细胞。接下来，用200微升的新鲜冰冷2%FBS每井PBS洗三次，每次洗净。最后洗涤后，将细胞颗粒重新在200微升新鲜冰冷2%FBS中重新暂停，每井PBS。对于流式细胞学采集和分析，立即将细胞加载到流式细胞仪上，并使用无感染红细胞的孔的线性前进和线性侧散射图来门控 THP-1 细胞。在此门中获取 10，000 个事件，并使用 THP-1 细胞与受感染的红细胞进行正对，以建立一个直方图，以测量溴化乙基荧光强度。为了进行分析，打开没有感染红细胞的孔的线性前进和线性侧散射图，并打开 THP-1 细胞。然后，在 FL-3 直方图中设置一个正门，并复制这些门到所有其他样品井上，以确定溴化乙基阳性 THP-1 细胞的百分比。对于每个测试样本，吞噬症可以报告为绝对值，也可以报告为相对吞噬症，使用正对比作为最大值计算为百分比。THP-1细胞应定期检查FC伽马受体表面表达通过流细胞仪。单元格对 CD16 应为负数，CD32 和 CD64 为正值。在这个结果化实验中，THP-1细胞首先根据其前向和侧散射特征进行门控，以便量化溴化乙基标记细胞的百分比，表明至少一个抗体蛋白分感染红细胞的细胞已经化为一个。负对样本应在FL3通道中生成单个负峰，在溴化乙基标记中观察到的事件很少。因此，平均噬菌体值，无论是作为绝对乙基溴素阳性THP-1细胞，作为相对噬菌体百分比，应该非常低。相反，正对应产生两个峰值的痕迹，一个负峰和一个位于溴化乙基标记内的明显正峰和分离良好的峰。阳性控制平均吞噬病值通常最高，其次是疟疾暴露的女性池和疟疾暴露妇女对照。尽管此方法生成一致的结果，但已观察到调整线的斜率系数的偏差。因此，建议使用相对值，尤其是在无法在单个实验中运行所有示例的情况下。请务必提前仔细规划实验，同时准备THP-1细胞和寄生虫，仅使用纯度大于80%的营养细胞。此外，请务必始终包括适当的控制措施，以便评估实验质量，并方便数据分析。

Research

JoVE Journal

Immunology and Infection

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

选择恶性疟原虫</emCytoadhesion>寄生虫对人脑血管内皮细胞

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

科研

免疫与感染

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

一个<em&gt;在体外</em&gt;共感染模型研究<em&gt;恶性疟原虫</em人原发性单核细胞源性的免疫细胞-HIV-1相互作用

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

一个简单的协议介导的血小板聚集<em&gt;恶性疟原虫</em&gt;受感染的红细胞在一个资源匮乏的设置

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

分离<em&gt;恶性疟原虫</em&gt;晚期感染的红细胞由磁性装置

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

高产净化<em&gt;恶性疟原虫</em&gt;裂殖子适用于Opsonizing抗体检测

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

在啮齿动物疟原虫原虫的体内评估和裂殖子入侵流式细胞仪

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

方法调查小RNA和核糖体占用的调控作用<em&gt;恶性疟原虫</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

流式细胞仪为基础的检测方法的NK细胞功能的监控

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

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, ...