Name: assessing respiratory immune responses to haemophilus influenzae
Uploaded: 2021-06-29T13:00:00
Description: Watch this Scientific Journal Video about Assessing Respiratory Immune Responses to Haemophilus Influenzae at JoVE.com

作者要感谢Monash Health临床免疫学的工作人员对这项工作的帮助。

这项工作得到了莫纳什健康人类研究伦理委员会的批准。该协议遵循人类研究伦理委员会的指导方针。
1.抗原制剂
注意：三种不同的抗原制剂可用于评估对Hi的免疫反应。这些是1）亚细胞成分（通常来自细菌细胞壁）;2）灭活和灭活细菌;3）活细菌。在开始任何实验之前确定每种抗原制剂的使用。
<ol><li>亚细胞成分<ol><li>从来源获取亚细胞成分，包括商?...

<ol>
	<li>Smith-Vaughan, H. C., Sriprakash, K. S., Leach, A. J., Mathews, J. D., Kemp, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+genetic+diversity+of+Haemophilus+influenzae+type+b+compared+to+nonencapsulated+H.+influenzae+in+a+population+in+which+H.+influenzae+is+highly+endemic.">Low genetic diversity of Haemophilus influenzae type b compared to nonencapsulated H. influenzae in a population in which H. influenzae is highly endemic.</a> Infection and Immunity. 66, 3403-3409 (1998).</li><li>Murphy, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haemophilus+and+Moxarella+infections.">Haemophilus and Moxarella infections.</a> Harrisons Principles of Internal Medicine. 152, (2018).</li><li>King, P. T., Sharma, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lung+immune+response+to+nontypeable+haemophilus+influenzae+(lung+immunity+to+NTHi).">The lung immune response to nontypeable haemophilus influenzae (lung immunity to NTHi).</a> Journal of Immunology Research. , 706376 (2015).</li><li>Ahearn, C. P., Gallo, M. C., Murphy, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+on+persistent+airway+infection+by+non-typeable+Haemophilus+influenzae+in+chronic+obstructive+pulmonary+disease.">Insights on persistent airway infection by non-typeable Haemophilus influenzae in chronic obstructive pulmonary disease.</a> Pathogens and Disease. 75, 9 (2017).</li><li>Brinkmann, V., 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=Neutrophil+extracellular+traps+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303, 1532-1535 (2004).</li><li>Brinkmann, V., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps:+is+immunity+the+second+function+of+chromatin.">Neutrophil extracellular traps: is immunity the second function of chromatin.</a> Journal of Cell Biology. 198, 773-783 (2012).</li><li>Jorch, S. K., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+emerging+role+for+neutrophil+extracellular+traps+in+noninfectious+disease.">An emerging role for neutrophil extracellular traps in noninfectious disease.</a> Nature Medicine. 23, 279-287 (2017).</li><li>Boe, D. M., Curtis, B. J., Chen, M. M., Ippolito, J. A., Kovacs, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+traps+and+macrophages:+new+roles+for+the+versatile+phagocyte.">Extracellular traps and macrophages: new roles for the versatile phagocyte.</a> Journal of Leukocyte Biology. 97, 1023-1035 (2015).</li><li>Cheng, O. Z., Palaniyar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NET+balancing:+a+problem+in+inflammatory+lung+diseases.">NET balancing: a problem in inflammatory lung diseases.</a> Frontiers in Immunology. 4, 1 (2013).</li><li>Jacobs, D. M., Ochs-Balcom, H. M., Zhao, J., Murphy, T. F., Sethi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+airway+bacterial+colonization+patterns+and+species-specific+interactions+in+chronic+obstructive+pulmonary+disease.">Lower airway bacterial colonization patterns and species-specific interactions in chronic obstructive pulmonary disease.</a> Journal of Clinical Microbiology. 56, (2018).</li><li>Barenkamp, S. J., Munson, R. S., Granoff, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtyping+isolates+of+Haemophilus+influenzae+type+b+by+outer-membrane+protein+profiles.">Subtyping isolates of Haemophilus influenzae type b by outer-membrane protein profiles.</a> The Journal of Infectious Diseases. 143, 668-676 (1981).</li><li>Barenkamp, 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=Outer+membrane+proteins+and+lipopolysaccharides+of+nontypeable+Haemophilus+influenzae.">Outer membrane proteins and lipopolysaccharides of nontypeable Haemophilus influenzae.</a> The Journal of Infectious Diseases. 165, 181-184 (1992).</li><li>Johnston, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+growth+and+maintenance+of+Haemophilus+influenzae.">Laboratory growth and maintenance of Haemophilus influenzae.</a> Current Protocols in Microbiology. , (2010).</li><li>King, P. 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=Adaptive+immunity+to+nontypeable+Haemophilus+influenzae.">Adaptive immunity to nontypeable Haemophilus influenzae.</a> American Journal of Respiratory and Critical Care Medicine. 167, 587-592 (2003).</li><li>Coleman, H. N., Daines, D. A., Jarisch, J., Smith, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+defined+media+for+growth+of+Haemophilus+influenzae+strains.">Chemically defined media for growth of Haemophilus influenzae strains.</a> Journal of Clinical Microbiology. 41, 4408-4410 (2003).</li><li>King, P. T., Ngui, J., Gunawardena, D., Holmes, P. W., Farmer, M. W., Holdsworth, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+humoral+immunity+to+non-typeable+Haemophilus+influenzae.">Systemic humoral immunity to non-typeable Haemophilus influenzae.</a> Clinical &amp; Experimental Immunology. 153, 376-384 (2008).</li><li>King, P. 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=Nontypeable+Haemophilus+influenzae+induces+sustained+lung+oxidative+stress+and+protease+expression.">Nontypeable Haemophilus influenzae induces sustained lung oxidative stress and protease expression.</a> PLoS One. 10, 0120371 (2015).</li><li>Aaron, S. D., 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=Granulocyte+inflammatory+markers+and+airway+infection+during+acute+exacerbation+of+chronic+obstructive+pulmonary+disease.">Granulocyte inflammatory markers and airway infection during acute exacerbation of chronic obstructive pulmonary disease.</a> American Journal of Respiratory and Critical Care Medicine. 163, 349-355 (2001).</li><li>King, P. 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=Lung+T-cell+responses+to+nontypeable+Haemophilus+influenzae+in+patients+with+chronic+obstructive+pulmonary+disease.">Lung T-cell responses to nontypeable Haemophilus influenzae in patients with chronic obstructive pulmonary disease.</a> The Journal of Allergy and Clinical Immunology. 131, 1314-1321 (2013).</li><li>Tsujikawa, 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=Robust+cell+detection+and+segmentation+for+image+cytometry+reveal+th17+cell+heterogeneity.">Robust cell detection and segmentation for image cytometry reveal th17 cell heterogeneity.</a> Cytometry A. 95, 389-398 (2019).</li><li>Sharma, R., O'Sullivan, K. M., Holdsworth, S. R., Bardin, P. G., King, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+macrophage+extracellular+traps+using+confocal+microscopy.">Visualizing macrophage extracellular traps using confocal microscopy.</a> Journal of Visualized Experiments: JoVE. (128), e56459 (2017).</li><li>Stiefel, P., Schmidt-Emrich, S., Maniura-Weber, K., Ren, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+aspects+of+using+bacterial+cell+viability+assays+with+the+fluorophores+SYTO9+and+propidium+iodide.">Critical aspects of using bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide.</a> BMC Microbiology. 15, 36 (2015).</li><li>Ueckert, J. E., Nebe von-Caron, G., Bos, A. P., ter Steeg, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+Lactobacillus+plantarum+to+monitor+lag+times,+cell+division+and+injury.">Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell division and injury.</a> Letters in Applied Microbiology. 25, 295-299 (1997).</li><li>Essilfie, A. 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=Combined+Haemophilus+influenzae+respiratory+infection+and+allergic+airways+disease+drives+chronic+infection+and+features+of+neutrophilic+asthma.">Combined Haemophilus influenzae respiratory infection and allergic airways disease drives chronic infection and features of neutrophilic asthma.</a> Thorax. 67, 588-599 (2012).</li><li>Huvenne, 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=Exacerbation+of+cigarette+smoke-induced+pulmonary+inflammation+by+Staphylococcus+aureus+enterotoxin+B+in+mice.">Exacerbation of cigarette smoke-induced pulmonary inflammation by Staphylococcus aureus enterotoxin B in mice.</a> Respiratory Research. 12, 69 (2011).</li><li>Radhakrishna, N., Farmer, M., Steinfort, D. P., King, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparison+of+Techniques+for+Optimal+Performance+of+Bronchoalveolar+Lavage.">A Comparison of Techniques for Optimal Performance of Bronchoalveolar Lavage.</a> Journal of Bronchology &amp; Interventional Pulmonology. 22, 300-305 (2015).</li><li>Quatromoni, J. G., Singhal, S., Bhojnagarwala, P., Hancock, W. W., Albelda, S. M., Eruslanov, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+disaggregation+method+for+human+lung+tumors+that+preserves+the+phenotype+and+function+of+the+immune+cells.">An optimized disaggregation method for human lung tumors that preserves the phenotype and function of the immune cells.</a> Journal of Leukocyte Biology. 97, 201-209 (2015).</li><li>Tighe, R. 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=Improving+the+quality+and+reproducibility+of+flow+cytometry+in+the+lung.+An+official+American+thoracic+society+workshop+report.">Improving the quality and reproducibility of flow cytometry in the lung. An official American thoracic society workshop report.</a> American Journal of Respiratory and Critical Care Medicine. 61, 150-161 (2019).</li><li>Yu, Y. 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=A+protocol+for+the+comprehensive+flow+cytometric+analysis+of+immune+cells+in+normal+and+inflamed+murine+non-lymphoid+tissues.">A protocol for the comprehensive flow cytometric analysis of immune cells in normal and inflamed murine non-lymphoid tissues.</a> PLoS One. 11, 0150606 (2016).</li><li>Duan, 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=Distinct+macrophage+subpopulations+characterize+acute+infection+and+chronic+inflammatory+lung+disease.">Distinct macrophage subpopulations characterize acute infection and chronic inflammatory lung disease.</a> Journal of Immunology. 189, 946-955 (2012).</li></ol>

这里列出的方法使用基于荧光的流式细胞术和共聚焦显微镜技术，可以结合使用以获得有关炎症性肺对Hi反应的详细信息。
建立要使用的Hi的适当抗原制剂至关重要，建议在这方面获得微生物学家的具体意见。Live Hi 会引起更强的反应，而杀死 Hi 准备和 Hi 组件更标准化，更容易存储。PI只会标记死细菌22;其他染料如羧基荧光素琥珀酰亚胺酯（CFSE）可用于标记...

流感嗜血杆菌 （Hi）是一种正常的共生细菌，存在于大多数健康成人的咽部。Hi可能具有多糖胶囊（A-F型，例如B型或HiB型）或缺乏胶囊并且不可分型（NTHi）1。这种细菌的粘膜定植始于儿童早期，并且存在不同定植菌株的更替2。这种细菌也能够侵入上呼吸道和下呼吸道;在这种情况下，它可以诱导免疫反应和炎症的激活3，4。这种炎症反应可能导致临床疾病并导致各种重要的呼吸系统疾病，包括鼻窦炎、中耳炎、支气管炎、囊性纤维化、肺炎和慢性阻塞性肺病 （COPD）。这些情况大多是由于NTHi菌株2引起的。本文将介绍使用流式细胞术和共聚焦显微镜评估对Hi的呼吸免疫反应的方法。
下面描述的方法已改编自成熟的技术，这些技术已被修改以评估对Hi的炎症反应。选择适当的抗原形式的Hi是该评估的关键部分。抗原制剂的范围从细胞壁成分到活细菌。为了建立和标准化检测，最初使用外周血样本可能非常有帮助。
流式细胞术能够在细胞水平上测量一个样品的各种参数和功能测定。与其他更通用的方法（例如酶联免疫吸附测定（ELISA）或ELISspot）相比，该技术的优点是可以评估特定的细胞反应（例如，活性氧（ROS）的产生或细胞内细胞因子的产生）。
细胞外陷阱由中性粒细胞（NET）5，6，7和其他细胞如巨噬细胞（MET）8表达。它们越来越被认为是一种关键的炎症反应，特别是在肺部感染中9.它们可以通过共聚焦荧光显微镜进行评估。该技术可以明确识别NET/MET，并将其表达与其他形式的细胞死亡区分开来6。
流式细胞术和共聚焦显微镜都是基于荧光的测定。它们的成功取决于生物样品的最佳过滤方案。这些方法确实需要一些时间来学习，并且需要适当的监督专业知识。所涉及的仪器的购买和运行成本也很高。使用它们的最佳环境包括主要大学和三级转诊医院。
本协议中使用的方法可转移用于研究涉及呼吸系统疾病的其他类似生物（例如， 卡他莫沙菌 和 肺炎链球菌）。NTHi还与其他常见的呼吸道细菌相互作用10。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ammonium chloride</td>
			<td>Sigma Aldrich</td>
			<td>213330</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin</td>
			<td>Sigma Aldrich</td>
			<td>B6542</td>
			<td></td>
		</tr>
		<tr>
			<td>CD28</td>
			<td>Thermofisher</td>
			<td>16-0289-81</td>
			<td></td>
		</tr>
		<tr>
			<td>CD49d</td>
			<td>Thermofisher</td>
			<td>534048</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI prolong gold</td>
			<td>Thermofisher</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>DHR123</td>
			<td>Sigma Aldrich</td>
			<td>109244-58-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Filcon sterile nylon mesh</td>
			<td>Becton Dickinson</td>
			<td>340606</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin substrate, Enzchek</td>
			<td>Molecular probes</td>
			<td>E12055</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS mix tube rotater</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-753</td>
			<td></td>
		</tr>
		<tr>
			<td>Medimachine</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td>Catalogue number not available</td>
		</tr>
		<tr>
			<td>Medicons 50 &#181;m</td>
			<td>Becton Dickinson</td>
			<td>340592</td>
			<td></td>
		</tr>
		<tr>
			<td>Pansorbin</td>
			<td>Sigma Aldrich</td>
			<td>507858</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma Aldrich</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma Aldrich</td>
			<td>8047152</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost slides</td>
			<td>Thermofisher</td>
			<td>11562203</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing respiratory immune responses to haemophilus influenzae

流感嗜血杆菌 （Hi）是一种普遍存在于一系列呼吸系统疾病中的细菌。可以使用各种不同的测定/技术来评估对该细菌的呼吸道免疫/炎症反应。流式细胞术和共聚焦显微镜是基于荧光的技术，可以详细表征生物反应。可以使用不同形式的Hi抗原，包括细胞壁成分，杀死/灭活制剂和活细菌。Hi是一种苛刻的细菌，需要富集培养基，但通常很容易在标准实验室环境中生长。Hi刺激的组织样本可以从外周血、支气管镜检查或切除的肺（例如，在接受肺癌手术治疗的患者中）获得。可以使用流式细胞术全面评估巨噬细胞和中性粒细胞功能，并测量各种参数，包括吞噬作用、活性氧和细胞内细胞因子产生。淋巴细胞功能（例如，T细胞和NK细胞功能）可以使用流式细胞术进行特异性评估，主要用于细胞内细胞因子的产生。Hi感染是中性粒细胞（NET）和巨噬细胞（MET）产生细胞外陷阱的有效诱导剂。共聚焦显微镜可以说是评估NET和MET表达的最佳方法，也可用于评估蛋白酶活性。肺对 流感嗜血杆菌 的免疫力可以使用流式细胞术和共聚焦显微镜进行评估。

代表性结果表明如何通过流式细胞术和共聚焦显微镜评估/定量对NTHi的炎症免疫反应。解释结果的一个关键部分是对照样品和受激发样品之间的荧光比较。通常需要进行一些初步实验来优化样品的染色。可以同时检测多少种不同的颜色取决于流式细胞仪/共聚焦显微镜上可用的通道数。结果显示用于评估1）ROS产生，2）人肺组织的细胞内细胞因子染色和3） 原位 酶图测量肺蛋白水解。
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Watch this Scientific Journal Video about Assessing Respiratory Immune Responses to Haemophilus Influenzae at JoVE.com

Assessing Respiratory Immune Responses to Haemophilus Influenzae

流感嗜血杆菌 诱发呼吸道炎症。本文将重点介绍如何使用流式细胞术和共聚焦显微镜来确定吞噬细胞和淋巴细胞对该细菌的免疫反应。

评估对流感嗜血杆菌的呼吸道免疫应答

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

流感嗜血杆菌是各种慢性肺部疾病（包括COPD和肺炎）炎症的主要原因。该协议描述了评估嗜血杆菌对肺部炎症影响的方法。该技术的优点是可以全面评估先天性和适应性免疫反应。将 450 微升外周血与 50 微升标记为流感嗜血杆菌的活化碘化丙啶在 5 毫升管中在 37 摄氏度的水浴中孵育 20 分钟。从水浴中取出样品，加入五微升DHR并涡旋10秒钟。然后将其放回水浴中再放置 10 分钟。从水浴中取出样品后，用五毫升0.8%氯化铵溶液裂解红细胞，并如文本中所述在流式细胞仪上分析样品。将外周血样本分成等分试样进行对照和抗原刺激。向两个样品添加共刺激抗体。然后将非典型流感嗜血杆菌添加到抗原样品中，并在37摄氏度和5%二氧化碳下孵育一小时。接下来，将高尔基体封闭剂布雷菲尔丁A添加到样品中，并再孵育五个小时。如前所述裂解红细胞后，使用 500 微升 1% 至 2% 的副甲醛固定白细胞一小时。使用血细胞计数器计数细胞，然后用100微升0.1%皂苷透化100万个细胞15分钟。接下来，用适当的荧光标记抗体孵育细胞。洗涤细胞，然后使用流式细胞仪进行分析。通过获取相关的淋巴细胞群来确定抗原反应细胞的比例。对未受刺激的细胞进行背景染色，以分析所有要分析的细胞因子。将大约 20 到 40 克的肺叶切除术样本切成三到五立方毫米的部分。将它们放入无菌的50微升腔室中，并使用适当的解聚器机械地碎片组织。组织解聚后，如图所示裂解红细胞，并将细胞重悬于无菌RPMI中。然后通过100微米无菌尼龙网过滤细胞，并使用台盼蓝排除方法计数活细胞。对于感染测定，将肺细胞重悬于RPMI中，最终浓度为每管每毫升400万个细胞。然后以每个细胞100个细菌的MOI感染细胞。松开盖子旋转半圈，以允许气体在管中转移。将细胞放入试管旋转器中，并在 37 摄氏度下孵育它们，同时以 12 RPM 旋转。刺激后一小时，加入格雷菲尔丁A以防止细胞因子的细胞外输出，并将细胞悬液再孵育16至22小时。第二天，用含有1%牛血清白蛋白和0.01%叠氮化钠的500微升PBS洗涤细胞悬液。接下来，对细胞悬液染色特定的人淋巴细胞表面标志物一小时。然后用PBS洗涤细胞并如前所述对其进行固定和透化。接下来，用细胞内细胞因子染色抗体孵育细胞一小时。然后洗涤细胞并将其重悬于100微升PBS中，然后在流式细胞仪上采集数据。通过金属蛋白酶的作用测量蛋白水解，将荧光素标记的明胶底物直接添加到肺组织切片载玻片上。将载玻片水平放置，并在37摄氏度的避光加湿室中孵育一小时。为了制备阴性对照，向切片中加入一个不含荧光明胶的反应缓冲液以进行进一步分析。使用向前和侧面分散的外周血单核细胞进行门控，以定义吞噬细胞群。吞噬细胞群通过CD14表达进一步定义以标记单核细胞。通过氧化DHR 123进行ROS测量以产生荧光。将受激发样品的中位数荧光与对照进行比较。使用流式细胞术测量淋巴细胞产生的细胞内细胞因子。首先分析细胞白细胞标志物CD45的表达，然后分析CD三和CD四，CD八。将CD三、CD四阳性细胞评估对照和刺激样品中细胞内细胞因子的产生情况。在未固定的肺组织切片中通过NC两个酶谱测量蛋白酶活性。荧光染色表明存在MMP活性，MMP活性也与染色质的表达共定位。向肺组织样品中添加抗体需要浓缩，细胞染色需要在初步实验中进行优化。肺组织样品的上清液可以使用ELISA等技术进一步分析其他炎症介质，这些技术可用于评估潜在疗法对肺部炎症的影响。

Research

JoVE Journal

Immunology and Infection

Assessing Respiratory Immune Responses to Haemophilus Influenzae

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

Seven Steps to Stellate Cells

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

呼吸爆发的响应为先天免疫健康的斑马鱼中的指标量化

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

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 ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

一个<em&gt;体外</em&gt;模型在HIV合并感染的背景下测量免疫应答疟疾

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

的气管内管理<em&gt;流感嗜血杆菌</em&gt;在小鼠模型来研究气道炎症

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...