Name: opsonophagocytic killing assay to assess immunological responses against bacterial pathogens
Uploaded: 2019-04-05T14:00:00
Description: Watch this Scientific Journal Video about Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens at JoVE.com

我们感谢穆恩·纳姆博士 (伯明翰阿拉巴马大学) 在我们的实验室建立 OPKA 检测方面提供的宝贵协助。这项工作得到了国家卫生研究院1R1AI123383-01A1 至 FYA 的支持。

1. HL-60 细胞的培养、分化和验证

<ol><li>用 l-谷氨酰胺和50毫升热灭活的胎儿牛血清制备由500毫升 RPMI 组成的 HL-60 细胞培养培养基。不要添加抗生素, 因为这可能会影响 HL-60 细胞的分化。</li>
	<li>为了 HL-60 细胞的繁殖/维持, 在37°c 和 5% co 2 的75厘米2通风瓶中, 在 hl-60 细胞培养基 10 毫升中培养 5 x 10 6 细胞.每3-4天通过一次细胞, 以保持最佳的细胞浓度。 请注意:...

<ol>
	<li>Romero-Steiner, 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=Standardization+of+an+opsonophagocytic+assay+for+the+measurement+of+functional+antibody+activity+against+Streptococcus+pneumoniae+using+differentiated+HL-60+cells.">Standardization of an opsonophagocytic assay for the measurement of functional antibody activity against Streptococcus pneumoniae using differentiated HL-60 cells.</a> Clinical and Diagnostic Laboratory Immunology. 4 (4), 415-422 (1997).</li><li>Nanra, J. 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=Capsular+polysaccharides+are+an+important+immune+evasion+mechanism+for+Staphylococcus+aureus.">Capsular polysaccharides are an important immune evasion mechanism for Staphylococcus aureus.</a> Human Vaccines and Immunotherapeutics. 9 (3), 480-487 (2013).</li><li>Ishibashi, K., Yamaguchi, O., Shiraiwa, Y., Ogihara, M., Shigeta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+therapy+of+Pseudomonas+aeruginosa+pyelonephritis+in+neutropenic+mice+with+human+antilipopolysaccharide+monoclonal+antibody+and+cefsulodin.">Combination therapy of Pseudomonas aeruginosa pyelonephritis in neutropenic mice with human antilipopolysaccharide monoclonal antibody and cefsulodin.</a> Journal of Urology. 155 (6), 2094-2097 (1996).</li><li>Middleton, D. R., Paschall, A. V., Duke, J. A., Avci, F. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+Hydrolysis+of+Pneumococcal+Capsular+Polysaccharide+Renders+the+Bacterium+Vulnerable+to+Host+Defense.">Enzymatic Hydrolysis of Pneumococcal Capsular Polysaccharide Renders the Bacterium Vulnerable to Host Defense.</a> Infection and Immunity. , (2018).</li><li>Baker, C. J., Noya, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+use+of+intravenous+immune+globulin+for+group+B+streptococcal+infection.">Potential use of intravenous immune globulin for group B streptococcal infection.</a> Reviews of Infectious Diseases. 12, 476-482 (1990).</li><li>Tian, H., Weber, S., Thorkildson, P., Kozel, T. R., Pirofski, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+opsonic+and+nonopsonic+serotype+3+pneumococcal+capsular+polysaccharide-specific+monoclonal+antibodies+against+intranasal+challenge+with+Streptococcus+pneumoniae+in+mice.">Efficacy of opsonic and nonopsonic serotype 3 pneumococcal capsular polysaccharide-specific monoclonal antibodies against intranasal challenge with Streptococcus pneumoniae in mice.</a> Infection and Immunity. 77 (4), 1502-1513 (2009).</li><li>Parameswarappa, S. 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=A+Semi-synthetic+Oligosaccharide+Conjugate+Vaccine+Candidate+Confers+Protection+against+Streptococcus+pneumoniae+Serotype+3+Infection.">A Semi-synthetic Oligosaccharide Conjugate Vaccine Candidate Confers Protection against Streptococcus pneumoniae Serotype 3 Infection.</a> Cell Chemical Biology. 23 (11), 1407-1416 (2016).</li><li>Jackson, 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=Randomized+clinical+trial+of+a+single+versus+a+double+dose+of+13-valent+pneumococcal+conjugate+vaccine+in+adults+55+through+74+years+of+age+previously+vaccinated+with+23-valent+pneumococcal+polysaccharide+vaccine.">Randomized clinical trial of a single versus a double dose of 13-valent pneumococcal conjugate vaccine in adults 55 through 74 years of age previously vaccinated with 23-valent pneumococcal polysaccharide vaccine.</a> Vaccine. 36 (5), 606-614 (2018).</li><li>Cywes-Bentley, 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=Antibody+to+a+conserved+antigenic+target+is+protective+against+diverse+prokaryotic+and+eukaryotic+pathogens.">Antibody to a conserved antigenic target is protective against diverse prokaryotic and eukaryotic pathogens.</a> Proceedings of the National Academy of Sciences of the USA. 110 (24), 2209-2218 (2013).</li><li>Fleck, R. A., Romero-Steiner, S., Nahm, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+HL-60+cell+line+to+measure+opsonic+capacity+of+pneumococcal+antibodies.">Use of HL-60 cell line to measure opsonic capacity of pneumococcal antibodies.</a> Clinical and Diagnostic Laboratory Immunology. 12 (1), 19-27 (2005).</li><li>Collins, S. J., Ruscetti, F. W., Gallagher, R. E., Gallo, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terminal+differentiation+of+human+promyelocytic+leukemia+cells+induced+by+dimethyl+sulfoxide+and+other+polar+compounds.">Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds.</a> Proceedings of the National Academy of Sciences of the USA. 75 (5), 2458-2462 (1978).</li><li>Melnick, N., Rajam, G., Carlone, G. M., Sampson, J. S., Ades, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+novel+therapeutic+approach+to+treating+severe+pneumococcal+infection+using+a+mouse+model.">Evaluation of a novel therapeutic approach to treating severe pneumococcal infection using a mouse model.</a> Clinical and Vaccine Immunology. 16 (6), 806-810 (2009).</li><li>Dwyer, M., Gadjeva, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opsonophagocytic+assay.">Opsonophagocytic assay.</a> Methods in Molecular Biology. 1100, 373-379 (2014).</li><li>Burton, R. L., Nahm, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+fourfold+multiplexed+opsonophagocytosis+assay+for+pneumococcal+antibodies+against+additional+serotypes+and+discovery+of+serological+subtypes+in+Streptococcus+pneumoniae+serotype+20.">Development of a fourfold multiplexed opsonophagocytosis assay for pneumococcal antibodies against additional serotypes and discovery of serological subtypes in Streptococcus pneumoniae serotype 20.</a> Clinical and Vaccine Immunololgy. 19 (6), 835-841 (2012).</li><li>Clarke, M. 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=Low-cost,+high-throughput,+automated+counting+of+bacterial+colonies.">Low-cost, high-throughput, automated counting of bacterial colonies.</a> Cytometry Part A. 77 (8), 790-797 (2010).</li><li>Aanei, C. 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=Database-guided+Flow-cytometry+for+Evaluation+of+Bone+Marrow+Myeloid+Cell+Maturation.">Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation.</a> Journal of Visualized Experiments. (141), e57867 (2018).</li><li>Hirz, T., Dumontet, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+Isolation+and+Analysis+to+Determine+their+Role+in+Lymphoma+Cell+Sensitivity+to+Therapeutic+Agents.">Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents.</a> Journal of Visulaized Experiments. (109), e53846 (2016).</li><li>Rieger, A. M., Nelson, K. L., Konowalchuk, J. D., Barreda, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+annexin+V/propidium+iodide+apoptosis+assay+for+accurate+assessment+of+cell+death.">Modified annexin V/propidium iodide apoptosis assay for accurate assessment of cell death.</a> Journal of Visualized Experiments. (50), 2597 (2011).</li><li>Blair, O. C., Carbone, R., Sartorelli, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+HL-60+promyelocytic+leukemia+cells:+simultaneous+determination+of+phagocytic+activity+and+cell+cycle+distribution+by+flow+cytometry.">Differentiation of HL-60 promyelocytic leukemia cells: simultaneous determination of phagocytic activity and cell cycle distribution by flow cytometry.</a> Cytometry. 7 (2), 171-177 (1986).</li><li>Middleton, D. 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=Identification+and+characterization+of+the+Streptococcus+pneumoniae+type+3+capsule-specific+glycoside+hydrolase+of+Paenibacillus+species+32352.">Identification and characterization of the Streptococcus pneumoniae type 3 capsule-specific glycoside hydrolase of Paenibacillus species 32352.</a> Glycobiology. 28 (2), 90-99 (2018).</li></ol>

Opka 在评估疫苗6,8引起的抗体介导的免疫反应中发挥着至关重要的作用。这种简化的 OPKA 的主要意义是在需要测试的条件 (即抗体、酶处理等) 中的适应性。从这个意义上说, 虽然这种检测方法可以用来测试视黄素 (即抗体) 在吞噬中的贡献, 但也可以用来评估如何克服通常抑制吞噬途径的毒力因素 (即胶囊多糖)。最大限度地减少多路复用 OPKA 中通常使用的...

光吞噬杀灭试验 (OPKA) 是将细菌结构或功能的改变与随后免疫反应和功能的变化联系起来的重要工具。因此, 它经常被用作一种补充检测方法, 以确定抗体治疗、候选疫苗、酶优化等基于免疫的疗效。虽然体内检测是确定细菌感染模型中有效清除或保护的必要条件, 但 OPKA 可用于评估细菌、免疫细胞和实验等最基本成分对细菌细胞死亡的免疫贡献治疗。以往的研究表明, opka 可以被修改和用于各种细菌和血清型, 包括肺炎链球菌1, 金黄色葡萄球菌 2, 铜绿假单胞菌3。此外, 这些优化的检测方法可用于评估不同的实验治疗方法, 包括酶使细菌更容易接触补充介导的免疫细胞4和抗体治疗以改善抗体的能力光圣化5。传统上, opka 检测已成功应用于基础和临床研究环境中, 作为由病原体特异性抗体6、7、8、9诱导的有力保护指标.
不同类型的免疫细胞可用于评估声超核细胞的杀伤。一个常用的吞噬种群是 HL-60 人白血病细胞系。该细胞系可作为培养中的灭活早幼粒细胞保存;然而, 他们可以通过不同的药物治疗区分成不同的激活状态10,11。用 N、n-二甲基甲酰胺处理 HL60 可将细胞系分化为具有较强吞噬活性的活化中性粒细胞11。虽然 HL-60 细胞已得到优化, 并经常用于这些吞噬检测 10, 其他原代多核白细胞可作为免疫臂的实验 12。
此外, 这些检测可以简化13或多路复用14 , 以查看要测试的细菌的多种抗生素耐药菌株。通过开发能够有效计算琼脂板15号单点细菌菌落形成单元 (Cfu) 的软件, 使多路复用方法更加可行.在这里, 我们描述了一种流线型的方法, 使用一种细菌菌株, hl-60 细胞, 小兔补充, 和血琼脂板。通过这种方法, 可以快速评估多种治疗方法, 以解决如何调节细菌感染的先天免疫反应的具体研究问题。

<table border="0" cellpadding="0" cellspacing="0" style="width:636px;" width="637">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Annexin V (APC conjugated)</td>
			<td style="width:147px;">BioLegend</td>
			<td style="width:119px;">640919</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">anti-CD35, human (PE conjugated)</td>
			<td style="width:147px;">BioLegend</td>
			<td style="width:119px;">333405</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">anti-CD71, human (PE conjugated)</td>
			<td style="width:147px;">BioLegend</td>
			<td style="width:119px;">334105</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:204px;">bacterial strain to be used (ie, Streptococcus pneumoniae, WU2)</td>
			<td style="width:147px;">Bacterial Respiratory Reference Laboratory (Dr. Moon Nahm)&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">blood agar plates</td>
			<td>Hardy Diagnostic</td>
			<td>A10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Fetal Clone serum</td>
			<td>HyClone</td>
			<td>SH30080.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">glycerol</td>
			<td>Sigma</td>
			<td>G9012-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">HL-60 cells</td>
			<td>ATCC</td>
			<td>CCL-240</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">IgG Isotype Control (PE conjugated)</td>
			<td style="width:147px;">BioLegend</td>
			<td style="width:119px;">400907</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">N,N-dimethylformamide (DMF)</td>
			<td>Fisher Chemical</td>
			<td>UN2265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">propidium iodide</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:119px;">P4864</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">RPMI media with L-glutamine</td>
			<td>Corning</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
	</tbody>
</table>

opsonophagocytic killing assay to assess immunological responses against bacterial pathogens

对宿主细菌定植的免疫反应的一个关键方面是吞噬。声嗜肺细胞杀伤试验 (OPKA) 是一种实验性方法, 其中吞噬细胞与细菌单位共同培养。免疫细胞会吞噬细胞, 以一种与互补有关的方式杀死细菌培养物。免疫介导的细胞杀伤效率取决于许多因素, 可以用来确定不同的细菌培养物在抵抗细胞死亡方面的比较。通过这种方式, 可以评估潜在的免疫疗法的有效性, 以适应特定的细菌菌株和/或血清型。在该协议中, 我们描述了一个简化的 OPKA, 利用基本的培养条件和细胞计数来确定细菌细胞的活力后, 共同培养的治疗条件和 HL-60 免疫细胞。该方法已成功地应用于多种肺炎球菌血清型、囊囊株和囊状菌株及其他细菌种类。该 OPKA 协议的优点是其简单、多功能 (因为此检测方法不限于作为视黄素的抗体处理), 以及最大限度地减少时间和试剂以评估基本实验组。

在启动 OPKA 之前, 应进行 HL-60 分化的验证。这可以通过流式细胞仪来确定 CD11b、CD35、CD71 和附件素 v 的细胞外表达 (图 1)。碘化钾也可作为一种生存能力的标记。用 DMF 治疗3天后, CD35 的表达应增加 (≥55% 的所有细胞), CD71 的表达应减少 (≤20% 的所有细胞)。环素 V + 和碘化丙酯 (PI +) 细胞的百分比应为 & lt;35, 以确保足够的细胞活力。如果这些百分比不符合最低要求, 则应按讨论...

Watch this Scientific Journal Video about Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens at JoVE.com

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

这种光吞噬杀灭方法用于比较吞噬免疫细胞根据不同的处理方法和条件对细菌做出反应和杀灭细菌的能力。传统上, 这种检测方法是评估针对视黄素细菌产生的抗体的效应功能的黄金标准。

评估细菌病原体免疫反应的声吞噬杀灭试验

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

该测定将细菌结构和功能的调制与免疫细胞为基础的抗菌活性相关。因此，在确定改善细菌清除的可能性时，可以评估潜在治疗的关键免疫治疗。蛋白心细胞杀菌分析传统上是评估对细菌产生的有效功能作为异音的金标准。与现有的高吞吐量标准化检测结果比较，该协议提供了简单性和多功能性。在该协议中，我们研究药物治疗对链球菌肺炎的影响，以及这些影响如何转化为细菌的噬菌体病的改善。在这里，我们应用OPKA到肺炎链球菌细胞。然而，此协议可以通过评估噬菌体杀死其他病原体的免疫细胞。投入时间优化细菌库存，确保最终的C比值在最佳范围内。我们强烈建议使用流式细胞学，以确保HL60细胞是可行的和活跃的，并尝试不同的培养条件，以确保功能活性细胞。可视化演示将更好地了解如何镀样以及如何实现可计数样本。首先，准备由500毫升RPMI组成的HL60细胞培养基，辅以L-谷氨酰胺和15毫升热灭活胎儿牛血清。为了殖和维持HL60细胞，在75平方厘米的75平方厘米的75平方厘米的75平方厘米的细胞培养500万细胞，在37摄氏度和5%的二氧化碳中。为了产生HL60细胞的工作库存，在HL60培养基中每毫升添加大约100万个细胞，并辅以10%二甲基硫化物到一毫升低温管中。为了分化HL60细胞，在HL60细胞培养基的15毫升培养基中培养1.5倍至7个细胞，在OPKA之前的三天内，无菌过滤器中补充0.6%DMF，在75平方厘米的烧瓶中，在37摄氏度和5%的二氧化碳中。要准备细菌库存样本，在37摄氏度下将 WU2 细菌菌株在适当的肉汤中生长约 2 到 4 小时。接下来，在六千次G下离心，将细菌分粒两分钟，并在适当的汤中将细胞重新在10至30毫升的15%甘油中重新储存。将细菌培养成无菌的1.5毫升离心管，储存在80摄氏度。接下来，在37摄氏度的水浴中解冻一瓶细菌。在无菌条件下，将细菌细胞取出并重新在500微升OBB中。在30摄氏度下，在血脂板上稀释未经处理的细菌存量，并培养板过夜。孵育步骤后，计算与HL60细胞共同培养的未经处理的细菌群每次稀释的菌落。记录细菌稀释产生最佳数量的可计数菌落为未来的OPKA与这种细菌库存。解冻一管细菌。在六千次G下培养细菌两分钟，并在上一步获得的最佳稀释时在OBB中重新暂停细胞颗粒。在圆底 96 井细胞培养板中，每井重新稀释的 10 微升的移液器。然后，将20微升的合适抗体或药物治疗添加到每个实验井重复。对于控制井，根据用于处理井的缓冲液使用 1XPBS 或 OBB。在室温下以大约 90 rpm 的转速摇动样品板一小时。要收获三天前用DF治疗的HL60分化细胞，在500次G下将细胞分粒3分钟。丢弃上一液，用至少 10 毫升 1XPBS 洗涤颗粒。在500次G下将洗净的细胞取出三分钟。丢弃上一液，重新暂停OBB中的细胞。以一到五个最终体积加入无菌未稀释的兔子小兔血清。经过一个小时的细菌培养，将每个样本分成两组重复的井。接下来，将 50 微升的 HL60 补充混合物添加到每组实验井中，仅向细菌井中加入 50 微升 OBB。在 37 摄氏度下摇动 96 井板一小时。要板样品，用 OBB 在 1 到 5 个最终体积稀释每一个井，使每个样品的体积至少为 50 微升。接下来，将每个样品的移液器50微升直接放到细菌培养板的指定区域，确保样品之间的足够间距。在室温下覆盖样品，让样品干燥约15分钟。在30摄氏度下一夜之间反转板块和文化。或者，厌氧罐中的培养板，以测试厌氧条件是否影响细菌生长或控制形态。经过隔夜培养后，计算每个指定样本区域中的菌落，然后通过将每个集中的活细胞数与相应的控件进行比较来分析数据。首次建立这种技术的最困难的方面可能是优化细菌库存的起始C比，以及确保HL60细胞得到有效分化。在启动OPKA之前，使用流式细胞学验证了HL60分化，并采用碘化二甲基作为可行性标记。用DMF治疗三天后，CD35的表达增加，CD71的表达减少。HL60治疗组中细菌CFO与相应非HL60治疗组相比的平均百分比用于比较不同治疗的细菌细胞存活率。结果表明，通过有效治疗，HL60共培养细胞与无HL60细胞之间的菌落数量不同，表明吞噬效率更高。结果表明，当细菌稀释未得到优化或电镀后未仔细观察菌群生长时，菌落的超生长会妨碍对菌落的准确计数。尝试此程序时，要记住的最重要的事情是仔细监测细菌孵化，以避免C比过度生长。体内测定可用于在宿主体内进行有效的细菌清除。这些测定可用于确认使用OPKA观察到的免疫功效可译至人类或动物模型。在以往的重大研究中，蛋白磷杀菌分析已经非常有效，将抗菌疗法与噬菌体免疫反应联系起来。使用细菌病原体可能是危险的。在检测过程中，请时刻佩戴防护个人装备。

Research

JoVE Journal

Immunology and Infection

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

酶联免疫斑点法（ELISPOT）：TH - 1细胞对微生物抗原的免疫应答的定量

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

科研

免疫与感染

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

抗原特异性在体内使用CFSE标记的靶细胞的杀伤含量

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

单克隆抗体的反对消极行政 H。 capsulatum和其他真菌病原体

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating ...

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

“发光法”的分类检测细菌病原体的记者噬菌体

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

斑马鱼的胚胎细胞内细菌病原体感染

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

饲养和注射<em&gt;烟草天蛾</em&gt;幼虫评估细菌的毒力

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Application of Long-term cultured Interferon-ÃƒÅ½Ã‚Â³ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...