Name: in vitro cellular activity evaluation of the nanoemulsion vaccine adjuvant ophiopogonin d
Uploaded: 2022-12-09T13:35:00
Description: Watch this Scientific Journal Video about In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D at JoVE.com

本研究获得国家重点研发计划2021YFC2302603号资助，国家自然科学基金项目第31670938、32070924、82041045、32000651号，重庆市自然科学基金项目第2014jcyjA0107号和2019jcyjA-msxmx0159号资助，北京市研究生科研与创新项目CYS21519号资助， 陆军军医大学专项拨款2020XBK24号，全国大学生创新创业计划202090031021号资助。

所有细胞实验均在配备基本手术室、缓冲室、无菌培养室以及鉴定和分析室的细胞工程实验室中进行。工作环境和条件没有微生物污染和其他有害因素。动物实验是根据《实验动物护理和使用指南》进行的，并得到第三军医大学实验动物福利与伦理委员会的批准。
1. 高压灭菌和材料制备
<ol><li>通过在121°C高压灭菌20分钟，通过湿热灭菌来制备试剂和消耗品，例?...

<ol>
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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=Ophiopogonin+D+attenuates+doxorubicin-induced+autophagic+cell+death+by+relieving+mitochondrial+damage+in+vitro+and+in+vivo.">Ophiopogonin D attenuates doxorubicin-induced autophagic cell death by relieving mitochondrial damage in vitro and in vivo.</a> The Journal of Pharmacology and Experimental Therapeutics. 352 (1), 166-174 (2015).</li><li>An, E. 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=Ophiopogonin+D+ameliorates+DNCB-induced+atopic+dermatitis-like+lesions+in+BALB/c+mice+and+TNF-alpha-+inflamed+HaCaT+cell.">Ophiopogonin D ameliorates DNCB-induced atopic dermatitis-like lesions in BALB/c mice and TNF-alpha- inflamed HaCaT cell.</a> Biochemical and Biophysical Research Communications. 522 (1), 40-46 (2020).</li><li>Song, X., 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=Effects+of+polysaccharide+from+Ophiopogon+japonicus+on+immune+response+to+Newcastle+disease+vaccine+in+chicken.">Effects of polysaccharide from Ophiopogon japonicus on immune response to Newcastle disease vaccine in chicken.</a> Pesquisa Veterin&#225;ria Brasileira. 36 (12), 1155-1159 (2016).</li><li>Tong, Y. N., 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=An+immunopotentiator,+ophiopogonin+D,+encapsulated+in+a+nanoemulsion+as+a+robust+adjuvant+to+improve+vaccine+efficacy.">An immunopotentiator, ophiopogonin D, encapsulated in a nanoemulsion as a robust adjuvant to improve vaccine efficacy.</a> Acta Biomaterialia. 77, 255-267 (2018).</li><li>Lin, C. 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=Hyaluronic+acid-glycine-cholesterol+conjugate-based+nanoemulsion+as+a+potent+vaccine+adjuvant+for+T+cell-mediated+immunity.">Hyaluronic acid-glycine-cholesterol conjugate-based nanoemulsion as a potent vaccine adjuvant for T cell-mediated immunity.</a> Pharmaceutics. 13 (10), 1569 (2021).</li><li>Xu, 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=Global+metabolomic+and+lipidomic+analysis+reveals+the+potential+mechanisms+of+hemolysis+effect+of+ophiopogonin+D+and+ophiopogonin+D'+in+vivo.">Global metabolomic and lipidomic analysis reveals the potential mechanisms of hemolysis effect of ophiopogonin D and ophiopogonin D' in vivo.</a> Chinese Medicine. 16 (1), 3 (2021).</li><li>Drane, D., Gittleson, C., Boyle, J., Maraskovsky, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ISCOMATRIX+adjuvant+for+prophylactic+and+therapeutic+vaccines.">ISCOMATRIX adjuvant for prophylactic and therapeutic vaccines.</a> Expert Review of Vaccines. 6 (5), 761-772 (2007).</li><li>Rudolf, 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=Microstructure+characterisation+and+identification+of+the+mechanical+and+functional+properties+of+a+new+PMMA-ZnO+composite.">Microstructure characterisation and identification of the mechanical and functional properties of a new PMMA-ZnO composite.</a> Materials. 13 (12), 2717 (2020).</li><li>Cannella, 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=Cytotoxicity+evaluation+of+endodontic+pins+on+L929+cell+line.">Cytotoxicity evaluation of endodontic pins on L929 cell line.</a> BioMed Research International. 2019, 3469525 (2019).</li><li>Jiao, 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=Limitations+of+MTT+and+CCK-8+assay+for+evaluation+of+graphene+cytotoxicity.">Limitations of MTT and CCK-8 assay for evaluation of graphene cytotoxicity.</a> RSC Advances. 5 (66), 53240-53244 (2015).</li><li>Ghasemi, M., Turnbull, T., Sebastian, S., Kempson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MTT+assay:+Utility,+limitations,+pitfalls,+and+interpretation+in+bulk+and+single-cell+analysis.">The MTT assay: Utility, limitations, pitfalls, and interpretation in bulk and single-cell analysis.</a> International Journal of Molecular Sciences. 22 (23), 12827 (2021).</li><li>Li, W., Zhou, J., Xu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+in+vitro+cytotoxicity+testing+of+medical+devices.">Study of the in vitro cytotoxicity testing of medical devices.</a> Biomedical Reports. 3 (5), 617-620 (2015).</li><li>Wu, F., 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=Correlation+between+elevated+inflammatory+cytokines+of+spleen+and+spleen+index+in+acute+spinal+cord+injury.">Correlation between elevated inflammatory cytokines of spleen and spleen index in acute spinal cord injury.</a> Journal of Neuroimmunology. 344, 577264 (2020).</li><li>Lewis, S. M., Williams, A., Eisenbarth, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+the+immune+system+in+the+spleen.">Structure and function of the immune system in the spleen.</a> Science Immunology. 4 (33), (2019).</li><li>Cox, J. H., Ferrari, G., Janetzki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cytokine+release+at+the+single+cell+level+using+the+ELISPOT+assay.">Measurement of cytokine release at the single cell level using the ELISPOT assay.</a> Methods. 38 (4), 274-282 (2006).</li><li>Elliott, A. 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亚单位疫苗具有极佳的安全性，但免疫原性较差。增强免疫原性的主要策略是物理吸附抗原或偶联佐剂，并将其纳入药物递送系统，以促进DC的摄取和呈递。 天然植物皂苷如奎莱皂苷及其衍生物剧毒，不适合开发人类疫苗17。因此，研究疫苗或佐剂对细胞的毒性作用是评价新疫苗的必要第一步。
本研究中提出的方案是根据ISO 10993-5：200918进?...

疫苗是预防和治疗传染病和非传染性疾病的重要手段。在疫苗制剂中适当添加佐剂和递送载体有利于增强抗原的免疫原性并产生持久的免疫应答1。除了经典的佐剂明矾（铝盐）外，目前上市的疫苗佐剂还有六种：MF592，3，AS04 3，AS03 3，AS01 3，CpG10184和Matrix-M5。一般来说，当人体遇到病毒攻击时，第一道和第二级防线（皮肤、粘膜、巨噬细胞）率先清除病毒，最后激活涉及免疫器官和免疫细胞的第三道防线。自 1920 年代初以来，铝盐和铝盐一直是人类疫苗中使用最广泛的佐剂，可引发有效的先天免疫反应6。然而，有人提出，经典佐剂激活抗原呈递细胞（APC），刺激免疫细胞产生特定的细胞因子和趋化因子，是佐剂起作用的机制，可能是佐剂仅对特异性免疫应答产生瞬时作用的原因之一7。有限的人用许可佐剂的存在是开发引起有效免疫反应的疫苗的限制因素8。
目前，越来越多的佐剂研究正在证明在小鼠中诱导强烈细胞免疫反应的能力。QS-21已被证明可诱导平衡的T辅助T-1（Th1）和T辅助2（Th2）免疫应答，产生更高水平的抗体滴度，并延长作为佐剂的保护，但其强毒性和溶血特性限制了其作为独立临床佐剂的发展9，10。OP-D（芦荟皂苷元-O-α-L-鼠李糖吡喃糖1-（1→2）-β-D-吡喃木糖基-（1→3）-β-D-呋喃糖苷）是从中药植物甾本科植物甾体皂苷4中分离出的甾体皂苷之一。此外，它是在黄桐中发现的主要药理活性成分（神麦山），已知具有一定的药理特性11。此外，它是百合科的成员，因其在细胞炎症和心肌损伤中的抑制和保护作用而被广泛使用。例如，OP-D改善了BALB / c小鼠中DNCB诱导的特应性皮炎样病变和肿瘤坏死因子α（TNF-α）炎性HaCaT细胞12。重要的是，OP-D通过减少活性氧的产生和破坏线粒体膜损伤，促进心血管系统的抗氧化保护，并保护心脏免受阿霉素诱导的自噬损伤。实验表明，服用OP-D和单桥粒苷有助于增强免疫健康，增加白细胞计数和DNA合成，并使抗体持续更长时间13。先前已发现OP-D具有佐剂作用14。
纳米乳液是由表面活性剂、油、助表面活性剂和水的组合组成的水包油纳米制剂12，15。这些纳米疫苗设计允许将抗原和佐剂封装在一起，以增强免疫刺激，保护抗原并促进树突状细胞（DC）成熟16。为了开发从筛选中获得的这些新型佐剂，重要的是找到适当的方法来评估它们的细胞反应能力。
该协议的目的是系统评估佐剂是否可以增强 体外 细胞培养中的吞噬作用和免疫细胞的表达，并详细说明主要的实验方法。实验分为四个小节：（1）OP-D和NOD对L929细胞的毒性通过细胞计数试剂盒-8（CCK-8）测定法确定;（2）通过脾细胞刺激和ELISpot测定检测免疫小鼠内分泌IFN-γ和IL-17A的细胞因子水平以及相应的细胞数;（3）使用共聚焦显微镜观察辅助刺激后DC的抗原呈递能力;（4）检测与佐剂共培养的正常小鼠腹膜巨噬细胞（PMs）获得的上清液中的三种细胞因子IL-1β，IL-6和TNF-α。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-EDTA (1x)</td>
			<td>GIBCO, USA</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well filter plates</td>
			<td>Millipore. Billerica, MA</td>
			<td>CLS3922</td>
			<td></td>
		</tr>
		<tr>
			<td>AlPO4</td>
			<td>General Chemical Company, USA</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>Countstar, China</td>
			<td>IC1000</td>
			<td></td>
		</tr>
		<tr>
			<td>BALB/c mice and C57BL/6 mice</td>
			<td>Beijing HFK Bioscience Co. Ltd</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>caprylic/capric triglyceride (GTCC)</td>
			<td>Beijing Fengli Pharmaceutical Co. Ltd., Beijing, China</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>CCK-8 kits</td>
			<td>Dojindo, Japan</td>
			<td>CK04</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counting Plate</td>
			<td>Costar, Corning, USA</td>
			<td>CO010101</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Sieve</td>
			<td>biosharp, China</td>
			<td>BS-70-CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810 R</td>
			<td>Eppendorf, Germany&#160;</td>
			<td>5811000398</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM basic(1x) medium</td>
			<td>GIBCO, USA</td>
			<td>C11885500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>DSZ5000X Inverted Microscope</td>
			<td>Nikon,Japan</td>
			<td>DSZ5000X</td>
			<td></td>
		</tr>
		<tr>
			<td>EL-35 (Cremophor-35)</td>
			<td>Mumbai, India</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISpot classic</td>
			<td>AID, Germany</td>
			<td>ELR06</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GIBCO, USA</td>
			<td>10099141C</td>
			<td></td>
		</tr>
		<tr>
			<td>Full-function Microplate Reader</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>VL0000D2</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>P42212</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Invitrogen, USA</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Granulocyte Macrophage Colony-Stimulating Factor</td>
			<td>GM-CSF, R&#38;D Systems, USA</td>
			<td>315-03</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Invitrogen, USA</td>
			<td>15630106</td>
			<td></td>
		</tr>
		<tr>
			<td>HF 90/240 Incubator</td>
			<td>Heal Force, Switzerland</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-4</td>
			<td>PeproTech, USA</td>
			<td>042149</td>
			<td></td>
		</tr>
		<tr>
			<td>L929 cell line</td>
			<td>FENGHUISHENGWU, China</td>
			<td>&#160;NCTC clone 929 (RRID:CVCL_0462)</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscopy</td>
			<td>Zeiss, Germany</td>
			<td>LSM 980</td>
			<td></td>
		</tr>
		<tr>
			<td>MONTANE 85 PPI</td>
			<td>SEPPIC, France</td>
			<td>L12910</td>
			<td></td>
		</tr>
		<tr>
			<td>MONTANOX 80 PPI</td>
			<td>SEPPIC, France</td>
			<td>36372K</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IFN-&#947; ELISA kit</td>
			<td>Dakewe, China</td>
			<td>1210002</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IFN-&#947; precoated ELISPOT kit</td>
			<td>Dakewe, China</td>
			<td>DKW22-2000-096</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-17A ELISA kit</td>
			<td>Dakewe, China</td>
			<td>1211702</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-17A ELISpotPLUS Kit</td>
			<td>ebiosciences, USA</td>
			<td>3521-4HPW-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-1&#946; ELISA kit</td>
			<td>Dakewe, China</td>
			<td>1210122</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-6 ELISA kit</td>
			<td>Dakewe, China</td>
			<td>1210602</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse TNF-&#945; ELISA kit</td>
			<td>Dakewe, China</td>
			<td>1217202</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids(100x)</td>
			<td>Invitrogen, USA</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophiopogonin-D</td>
			<td>Chengdu Purui Technology Co. Ltd</td>
			<td>945619-74-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Invitrogen, USA</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>Solarbio, China</td>
			<td>CA1620</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>ZSGB-BIO, China</td>
			<td>ZLI-9062</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer</td>
			<td>Solarbio, China</td>
			<td>R1010</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Hyclone (Life Technology), USA</td>
			<td>SH30809.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate(100 mM)</td>
			<td>Invitrogen, USA</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Squalene</td>
			<td>Sigma, USA</td>
			<td>S3626</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;- Mercaptoethanol</td>
			<td>Invitrogen, USA</td>
			<td>21985023</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro cellular activity evaluation of the nanoemulsion vaccine adjuvant ophiopogonin d

作为疫苗的主要成分，佐剂可以直接诱导或增强与抗原相关的强大、广泛、先天和适应性免疫应答。Ophiopogonin D（OP-D）是从植物 Ophiopogon japonicus中提取的纯化成分，已被发现可用作疫苗佐剂。采用低能乳化法制备纳米乳液蛇腓磷素D（NOD）可以有效克服OP-D溶解度低、毒性低的问题。在本文中，研究了一系列用于细胞活性评估的 体外 方案。 使用细胞计数试剂盒-8测定法测定L929的细胞毒性作用。然后，采用ELISA和ELISpot方法检测免疫小鼠脾细胞刺激培养后分泌的细胞因子水平和相应的免疫细胞数。此外，通过激光扫描共聚焦显微镜（CLSM）观察从C57BL / 6小鼠中分离并在与GM-CSF加IL-4孵育后成熟的骨髓来源树突状细胞（BMDCs）中的抗原摄取能力。重要的是，在用佐剂共培养空白小鼠的腹膜巨噬细胞（PM）24小时后，通过ELISA试剂盒测量IL-1β，IL-6和肿瘤坏死因子α（TNF-α）细胞因子的水平来确认巨噬细胞活化。希望该协议将为其他研究人员提供直接有效的实验方法来评估新型疫苗佐剂的细胞反应有效性。

佐剂OP-D和NOD的细胞活性评估根据方案在 体外 完成。L929成纤维细胞是NOD体 外 毒性测试的有用筛选模型（图1）。脾脏中炎性细胞因子水平的定量可以帮助研究人员更好地了解免疫反应（图2）。使用ELISpot监测CTL是在临床试验中评估抗原特异性T细胞免疫和筛选候选疫苗的金标准（图3）。DC对抗原摄取的增加可以引发增...

Watch this Scientific Journal Video about In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D at JoVE.com

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

该协议提供了评估纳米乳剂蛇腓苷D佐剂是否促进有效细胞免疫应答的详细方法。

体外 纳米乳剂疫苗佐剂蛇腓磷素D的细胞活性评价

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

该协议可以为其他研究人员提供直接有效的实验方法和详细步骤，以评估新型疫苗佐剂的细胞反应有效性。它不仅可以为研究人员提供佐剂的特定细胞评估方法，还可以分解BM病等提取方法。该协议不仅证明无毒性和高细胞递送，而且还为佐剂评估领域提供了详细的程序。首先，打开水浴并将温度调节到37摄氏度。然后从液氮中收集一管冷冻的L929细胞后，在水浴中快速解冻。将细胞移液到15毫升无菌离心管中后，加入两毫升DMEM并充分混合。将样品以129 x g 离心五分钟。弃去上清液后，加入两毫升DMEM重悬细胞。再次离心样品。弃去上清液后，加入6毫升DMEM完全培养基进行重悬，并转移到含有5%二氧化碳的37摄氏度培养箱中的T25平方厘米培养瓶中培养48小时。在初次免疫后的第24天，将小鼠从动物室中取出，将其放入玻璃盘中，并在75%酒精中浸泡五分钟。将离心管放在离心管架上，对一次性培养皿进行编号，并用10毫升移液管向每个培养皿中加入5毫升PBS。在小鼠左腹侧中间用剪刀做一个六到八厘米的切口。撕开皮肤，露出腹壁，找到脾脏的长红色条带。接下来，用镊子提起脾脏下侧的腹膜。切开并向上翻转以露出脾脏。用镊子提起脾脏。用眼科剪刀分离脾脏下方的结缔组织，取出脾脏。将脾脏放入含有五毫升PBS的培养皿中，并用筛子和注射器柱塞研磨。研磨后，按照编号将液体转移到带有移液管的15毫升离心管中。接下来，将液体以453 x g 离心五分钟。弃去上清液后，向每管中加入三毫升红细胞裂解缓冲液。重悬细胞并在室温下裂解10分钟。然后在每个试管中加入 10 至 12 毫升 PBS。混合并以453×g离心五分钟后，弃去上清液，向每个离心管中加入10毫升PBS并重悬细胞。在细胞计数板的孔中取20微升每个样品，并使用自动细胞计数器记录活细胞的数量。接下来，将样品以453 x g 离心五分钟。弃去上清液后，用RF10培养基稀释至每毫升2.5×10至第六个细胞，并以每孔100微升的速度加入96孔板中。用剪刀在小鼠腹部下方切一个六到八厘米的切口，夹住开口的两端，使其向不同方向分开，露出老鼠的腿。将小鼠股骨与小鼠身体分开，将胫骨与关节分开。并保持两端的骨头完好无损。接下来，用剪刀和镊子从股骨两端的关节中去除残留的组织和软骨。将股骨浸泡在 75% 酒精中五分钟，然后浸泡在无菌 PBS 溶液中以洗去表面酒精。然后用剪刀剪掉股骨的末端，并在无菌PBS溶液的无菌培养皿中冲洗骨髓，然后用一毫升注射器抽吸。重复洗涤三到五次。接下来，通过细胞筛过滤并将骨髓来源的树突状细胞收集到15毫升离心管中。将样品以290×g离心5分钟后，弃去上清液并加入4毫升红细胞裂解缓冲液，重悬并在室温下裂解5分钟。接下来，加入10毫升无菌PBS溶液以中和裂解物，并以290×g离心五分钟后，弃去上清液。将细胞重悬于含有1%青霉素 - 链霉素溶液和10%胎牛血清的一毫升DMEM中并计数。然后将粒细胞巨噬细胞集落刺激因子加白细胞介素-4加入培养基中。将细胞浓度调节至每毫升5 x 10至五分之一，并在盖玻片上接种细胞。将细胞悬液以每孔2毫升的速度加入六孔板后，将板置于37摄氏度的加湿培养箱中，用5%二氧化碳放置48小时。两天后完全更换培养基，四天后更换一半培养基。安乐死后将小鼠浸入75%酒精中，并按编号顺序将它们面朝上放入玻璃培养皿中。将小鼠通过转移窗口进入无菌手术室，并将它们放在手术台上五分钟。使用注射器吸出10毫升盐水。将小鼠向下倾斜约45度并注射到腹腔中间。每10毫升将约5毫升细胞悬液吸入15毫升离心管中，重复注射三次。将细胞悬液以129×g离心5分钟，以获得小鼠腹膜原代巨噬细胞。L929成纤维细胞是NOD体外毒性测试的有用筛选模型。量化脾脏中的炎性细胞因子水平可以帮助研究人员更好地了解免疫反应。使用酶联免疫斑点监测细胞毒性 T 淋巴细胞是在临床试验中评估抗原特异性 T 细胞免疫和筛选候选疫苗的金标准。树突状细胞对抗原摄取的增加可以引发增强的适应性免疫反应。巨噬细胞在向T细胞呈递抗原以及诱导其他抗原呈递细胞表达共刺激分子方面起着重要作用，从而启动适应性免疫反应。在药物与细胞作用的比例下分离细胞至关重要。

Research

JoVE Journal

Immunology and Infection

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

免疫与感染

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

在FDA批准的Vero细胞疫苗开发产生重组沙粒

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

限制性内切酶克隆方法，以评估<em&gt;在体外</em&gt; HIV-1 C亚型的Gag-MJ4嵌合病毒的复制能力

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

体外ELISA测试评估狂犬病疫苗效力

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits (Oryctolagus cuniculus)

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits Oryctolagus cuniculus

新西兰白兔产妇免疫研究中的内在免疫和牛奶收集

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of ...

体外 纳米乳剂疫苗佐剂蛇腓磷素D的细胞活性评价

体外纳米乳剂疫苗佐剂蛇腓磷素D的细胞活性评价