Name: isolation of low endotoxin content extracellular vesicles derived from cancer cell lines
Uploaded: 2023-02-17T13:30:00
Description: Watch this Scientific Journal Video about Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines at JoVE.com

这项工作得到了波兰国家科学中心的支持，批准号为2019/33/B/NZ5/00647。我们要感谢雅盖隆大学医学院分子医学微生物学系的Tomasz Gosiewski教授和Agnieszka Krawczyk教授在检测电动汽车中细菌DNA方面的宝贵帮助。

1. 超速离心管的制备
<ol><li>使用无菌一次性试管。如果无法做到这一点，请在使用无菌巴斯德移液管或其他一次性涂抹器用洗涤剂清洗后重复使用超速离心管。请记住，超速离心管应专用于一种类型的离心材料（细胞培养上清液/血清/血浆）和物种（人/小鼠/等）。</li>
	<li>洗涤剂洗涤后，用去离子的无LPS水冲洗超速离心管3次。 注意：请勿使用劣质水。</li>
	<li>干燥超速离心管，?...

<ol>
	<li>Th&#233;ry, 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=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).</li><li>Y&#225;&#241;ez-M&#243;, 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=Biological+properties+of+extracellular+vesicles+and+their+physiological+functions.">Biological properties of extracellular vesicles and their physiological functions.</a> Journal of Extracellular Vesicles. 4, 27066 (2015).</li><li>van Niel, 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=Challenges+and+directions+in+studying+cell-cell+communication+by+extracellular+vesicles.">Challenges and directions in studying cell-cell communication by extracellular vesicles.</a> Nature Reviews Molecular Cell Biology. 23 (5), 369-382 (2022).</li><li>Ngkelo, A., Meja, K., Yeadon, M., Adcock, I., Kirkham, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LPS+induced+inflammatory+responses+in+human+peripheral+blood+mononuclear+cells+is+mediated+through+NOX4+and+Gi&#945;+dependent+PI-3+kinase+signalling.">LPS induced inflammatory responses in human peripheral blood mononuclear cells is mediated through NOX4 and Gi&#945; dependent PI-3 kinase signalling.</a> Journal of Inflammation. 9 (1), (2012).</li><li>Schwarz, H., Schmittner, M., Duschl, A., Horejs-Hoeck, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+endotoxin+contaminations+in+recombinant+proteins+are+sufficient+to+activate+human+CD1c++dendritic+cells.">Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells.</a> PLOS ONE. 9 (12), 113840 (2014).</li><li>Zanoni, I., Granucci, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CD14+in+host+protection+against+infections+and+in+metabolism+regulation.">Role of CD14 in host protection against infections and in metabolism regulation.</a> Frontiers in Cellular and Infection Microbiology. 3, 32 (2013).</li><li>Takashiba, 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=Differentiation+of+monocytes+to+macrophages+primes+cells+for+lipopolysaccharide+stimulation+via+accumulation+of+cytoplasmic+nuclear+factor+kB.">Differentiation of monocytes to macrophages primes cells for lipopolysaccharide stimulation via accumulation of cytoplasmic nuclear factor kB.</a> Infection and Immunity. 67 (11), 5573-5578 (1999).</li><li>Schwarz, 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=Biological+activity+of+masked+endotoxin.">Biological activity of masked endotoxin.</a> Scientific Reports. 7, 44750 (2017).</li><li>Danesh, 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=Granulocyte-derived+extracellular+vesicles+activate+monocytes+and+are+associated+with+mortality+in+intensive+care+unit+patients.">Granulocyte-derived extracellular vesicles activate monocytes and are associated with mortality in intensive care unit patients.</a> Frontiers in Immunology. 9, 956 (2018).</li><li>Barry, O. P., Pratico, D., Savani, R. C., FitzGerald, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+monocyte-endothelial+cell+interactions+by+platelet+microparticles.">Modulation of monocyte-endothelial cell interactions by platelet microparticles.</a> The Journal of Clinical Investigation. 102 (1), 136-144 (1998).</li><li>Sadallah, S., Eken, C., Martin, P. J., Schifferli, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticles+(ectosomes)+shed+by+stored+human+platelets+downregulate+macrophages+and+modify+the+development+of+dendritic+cells.">Microparticles (ectosomes) shed by stored human platelets downregulate macrophages and modify the development of dendritic cells.</a> Journal of Immunology. 186 (11), 6543-6552 (2011).</li><li>Soekmadji, 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=The+future+of+Extracellular+Vesicles+as+Theranostics+-+an+ISEV+meeting+report.">The future of Extracellular Vesicles as Theranostics - an ISEV meeting report.</a> Journal of Extracellular Vesicles. 9 (1), 1809766 (2020).</li><li>Gioannini, T. 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=Isolation+of+an+endotoxin-MD-2+complex+that+produces+Toll-like+receptor+4-dependent+cell+activation+at+picomolar+concentrations.">Isolation of an endotoxin-MD-2 complex that produces Toll-like receptor 4-dependent cell activation at picomolar concentrations.</a> Proceedings of the National Academy of Sciences. 101 (12), 4186-4191 (2004).</li><li>Roslansky, P. F., Dawson, M. E., Novitsky, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastics,+endotoxins,+and+the+Limulus+amebocyte+lysate+test.">Plastics, endotoxins, and the Limulus amebocyte lysate test.</a> Journal of Parenteral Science and Technology. 45 (2), 83-87 (1991).</li><li>Fishel, S., Jackson, P., Webster, J., Faratian, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxins+in+culture+medium+for+human+in+vitro+fertilization.">Endotoxins in culture medium for human in vitro fertilization.</a> Fertility and Sterility. 49 (1), 108-111 (1988).</li><li>Schulz, E., Karagianni, A., Koch, M., Fuhrmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hot+EVs+-+How+temperature+affects+extracellular+vesicles.">Hot EVs - How temperature affects extracellular vesicles.</a> European Journal of Pharmaceutics and Biopharmaceutics. 5 (146), 55-63 (2020).</li><li>Osteikoetxea, 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=Differential+detergent+sensitivity+of+extracellular+vesicle+subpopulations.">Differential detergent sensitivity of extracellular vesicle subpopulations.</a> Organic &amp; Biomolecular Chemistry. 13 (38), 9775-9782 (2015).</li><li>Sakudo, A., Yagyu, Y., Onodera, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disinfection+and+sterilization+using+plasma+technology:+fundamentals+and+future+perspectives+for+biological+applications.">Disinfection and sterilization using plasma technology: fundamentals and future perspectives for biological applications.</a> International Journal of Molecular Sciences. 20 (20), 5216 (2019).</li><li>Shintani, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethylene+oxide+gas+sterilization+of+medical+devices.">Ethylene oxide gas sterilization of medical devices.</a> Biocontrol Science. 22 (1), 1-16 (2017).</li><li>Cvjetkovic, A., L&#246;tvall, J., L&#228;sser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+rotor+type+and+centrifugation+time+on+the+yield+and+purity+of+extracellular+vesicles.">The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles.</a> Journal of Extracellular Vesicles. 3, (2014).</li><li>Salamon, 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=Comparison+of+iSeq+and+MiSeq+as+the+two+platforms+for+16S+rRNA+sequencing+in+the+study+of+the+gut+of+rat+microbiome.">Comparison of iSeq and MiSeq as the two platforms for 16S rRNA sequencing in the study of the gut of rat microbiome.</a> Applied Microbiology and Biotechnology. 106 (22), 7671-7681 (2022).</li><li>Baj-Krzyworzeka, M., Szatanek, R., Weglarczyk, K., Baran, J., Zembala, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-derived+microvesicles+modulate+biological+activity+of+human+monocytes.">Tumour-derived microvesicles modulate biological activity of human monocytes.</a> Immunology Letters. 113 (2), 76-82 (2007).</li><li>Chaiwut, R., Kasinrerk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Very+low+concentration+of+lipopolysaccharide+can+induce+the+production+of+various+cytokines+and+chemokines+in+human+primary+monocytes.">Very low concentration of lipopolysaccharide can induce the production of various cytokines and chemokines in human primary monocytes.</a> BMC Research Notes. 15 (1), 42 (2022).</li><li>Van Deun, 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=The+impact+of+disparate+isolation+methods+for+extracellular+vesicles+on+downstream+RNA+profiling.">The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling.</a> Journal of Extracellular Vesicles. 3, 24858 (2014).</li><li>Lehrich, B. M., Liang, Y., Fiandaca, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foetal+bovine+serum+influence+on+in+vitro+extracellular+vesicle+analyses.">Foetal bovine serum influence on in vitro extracellular vesicle analyses.</a> Journal of Extracellular Vesicles. 10 (3), 12061 (2021).</li><li>Pham, C. 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=Bovine+extracellular+vesicles+contaminate+human+extracellular+vesicles+produced+in+cell+culture+conditioned+medium+when+'exosome-depleted+serum'+is+utilised.">Bovine extracellular vesicles contaminate human extracellular vesicles produced in cell culture conditioned medium when 'exosome-depleted serum' is utilised.</a> Archives of Biochemistry and Biophysics. 708, 108963 (2021).</li><li>Li, Y., Boraschi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxin+contamination:+a+key+element+in+the+interpretation+of+nanosafety+studies.">Endotoxin contamination: a key element in the interpretation of nanosafety studies.</a> Nanomedicine. 11 (3), 269-287 (2016).</li><li>&#193;lvarez, E., 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+system+dynamics+model+to+predict+the+human+monocyte+response+to+endotoxins.">A system dynamics model to predict the human monocyte response to endotoxins.</a> Frontiers in Immunology. 8, 915 (2017).</li><li>Busatto, 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=Tangential+flow+filtration+for+highly+efficient+concentration+of+extracellular+vesicles+from+large+volumes+of+fluid.">Tangential flow filtration for highly efficient concentration of extracellular vesicles from large volumes of fluid.</a> Cells. 7 (12), 273 (2018).</li><li>Ga&#322;uszka, 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=Transition+metal+containing+particulate+matter+promotes+Th1+and+Th17+inflammatory+response+by+monocyte+activation+in+organic+and+inorganic+compounds+dependent+manner.">Transition metal containing particulate matter promotes Th1 and Th17 inflammatory response by monocyte activation in organic and inorganic compounds dependent manner.</a> International Journal of Environmental Research and Public Health. 17 (4), 1227 (2020).</li><li>Falagas, M. E., Kasiakou, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+polymyxins:+a+systematic+review+of+the+evidence+from+old+and+recent+studies.">Toxicity of polymyxins: a systematic review of the evidence from old and recent studies.</a> Critical Care. 10 (1), 27 (2006).</li><li>Petsch, D., Anspach, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxin+removal+from+protein+solutions.">Endotoxin removal from protein solutions.</a> Journal of Biotechnology. 76 (2-3), 97-119 (2000).</li></ol>

在过去几年中，正确分离电动汽车的方法变得越来越重要，使其能够进一步进行可靠的分析，例如，在获得可靠的组学和功能数据的背景下24。根据以前的研究经验，似乎不仅隔离方法的类型，而且该过程期间的其他条件都可能很重要。使用耗尽电动汽车的FBS被广泛认为是必需品25，26;然而，对电动汽车内毒素污染的监测往往被忽视。<...

根据MISEV 2018命名法，细胞外囊泡（EV）是一个统称，描述了细胞分泌的膜囊泡的各种亚型，这些囊泡在许多生理和病理过程中起着至关重要的作用1，2。此外，电动汽车有望成为各种疾病的新型生物标志物，以及治疗剂和药物输送载体。然而，充分发挥其潜力是困难的，因为与获取它们相关的技术障碍仍然存在许多3.其中一个挑战是分离无内毒素的电动汽车，这在许多情况下被忽视了。最常见的内毒素之一是脂多糖（LPS），它是革兰氏阴性细菌细胞壁的主要成分，由于各种细胞释放大量炎性细胞因子，可引起急性炎症反应4，5。LPS通过与LPS结合蛋白结合诱导反应，然后与骨髓细胞上的CD14 / TLR4 / MD2复合物相互作用。这种相互作用导致MyD88和TRIF依赖性信号通路的激活，进而触发核因子κB（NFkB）。NFkB易位到细胞核启动细胞因子的产生6。单核细胞和巨噬细胞对 LPS 高度敏感，暴露于 LPS 会导致炎症细胞因子和趋化因子（例如 IL-6、IL-12、CXCL8 和 TNF-α）的释放7，8。CD14结构能够结合具有相似亲和力的不同LPS物种，并作为其他toll样受体（TLR）（TLR1，2，3，4，6，7和9）的辅助受体6。关于EV对单核细胞/巨噬细胞影响的研究数量仍在增加9，10，11。特别是从研究单核细胞、其亚群和其他免疫细胞的功能的角度来看，内毒素的存在，甚至它们在EV中的掩盖存在非常重要12。被忽视的电动汽车内毒素污染可能会导致误导性结论并掩盖其真正的生物活性。换句话说，使用单核细胞需要对没有内毒素污染的信心13。内毒素的潜在来源可以是水、商业获得的培养基和血清、培养基成分和添加剂、实验室玻璃器皿和塑料器皿5，14，15。
因此，本研究旨在制定一种分离低内毒素电动汽车的方案。该协议提供了有关如何在EV分离过程中避免内毒素污染而不是从EV中去除内毒素的简单提示。以前，已经提出了许多关于如何从例如纳米医学中使用的工程纳米颗粒中去除内毒素的方案;然而，它们都对电动汽车等生物结构没有用。纳米颗粒的有效去热原可以通过乙醇或乙酸漂洗，在175°C加热3h，γ照射或Triton X-100处理进行;然而，这些程序导致电动汽车的破坏16，17。
所提出的协议是一项开创性研究，专注于避免 EV 中的内毒素杂质，这与之前关于 EV 对单核细胞影响的研究不同9. 将拟议的原则应用于实验室实践可能有助于获得可靠的研究结果，这在考虑电动汽车作为临床治疗剂的潜在用途时至关重要12。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;Alix (3A9) Mouse mAb&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>2171</td>
			<td></td>
		</tr>
		<tr>
			<td>1250ul Filter Universal Pipette Tips, Clear, Polypropylene, Non-Pyrogenic</td>
			<td>GoogLab Scientific</td>
			<td>GBFT1250-R-NS</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCanto II Flow Cytometr</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CBA Human Th1/Th2 Cytokine Kit II</td>
			<td>BD Biosciences</td>
			<td>551809</td>
			<td></td>
		</tr>
		<tr>
			<td>CD9 (D8O1A) Rabbit mAb</td>
			<td>Cell Signaling Technology</td>
			<td>13174</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc Imaging System</td>
			<td>Bio-Rad Laboratories, Inc.&#160;</td>
			<td>17001401</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#8217;s Modified Eagle&#8217;s Medium)&#160;</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>ELX800NB, Universal Microplate Reader</td>
			<td>BIO-TEK INSTRUMENTS, INC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum South America Ultra Low Endotoxin</td>
			<td>&#160;Biowest</td>
			<td>&#160;S1860-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin, 50 mg/mL</td>
			<td>&#160;PAN &#8211; Biotech</td>
			<td>P06-13100</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG- HRP</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG- HRP</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2005</td>
			<td></td>
		</tr>
		<tr>
			<td>Immun-Blot PVDF Membrane</td>
			<td>Bio-Rad Laboratories, Inc.&#160;</td>
			<td>1620177</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS from Salmonella abortus equi S-form (TLRGRADE)</td>
			<td>&#160;Enzo Life Sciences, Inc.</td>
			<td>ALX-581-009-L002</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Electrophoretic Transfer Cell</td>
			<td>Bio-Rad Laboratories, Inc.&#160;</td>
			<td>1703930</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoparticle Tracking Analysis&#160;</td>
			<td>Malvern Instruments Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample Buffer (4X)</td>
			<td>&#160;Invitrogen</td>
			<td>&#160;NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Sample Reducing Agent (10x)</td>
			<td>&#160;Invitrogen</td>
			<td>NP0004</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma Aldrich</td>
			<td>P7793</td>
			<td>transparent film</td>
		</tr>
		<tr>
			<td>Perfect 100-1000 bp DNA Ladder</td>
			<td>EURx</td>
			<td>E3141-01&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PierceTM Chromogenic Endotoxin Quant Kit</td>
			<td>Thermo Scientific</td>
			<td>A39552</td>
			<td></td>
		</tr>
		<tr>
			<td>PP Oak Ridge Tube with sealing caps</td>
			<td>Thermo Scientific</td>
			<td>3929, 03613</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>RPMI-1640 (Gibco)</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>SimpliAmp Thermal Cycler</td>
			<td>Applied Biosystem</td>
			<td>A24811</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall wX+ ULTRA SERIES Centrifuge with T-1270 rotor</td>
			<td>Thermo Scientific</td>
			<td>75000100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sub-Cell GT Horizontal Electrophoresis System</td>
			<td>Bio-Rad Laboratories, Inc.&#160;</td>
			<td>1704401</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Pico PLUS Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td>34577</td>
			<td></td>
		</tr>
		<tr>
			<td>SW480 cell line</td>
			<td>American Type Culture Collection(ATCC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SW480 cell line</td>
			<td>American Type Culture Collection (ATCC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter 0.22 um</td>
			<td>TPP</td>
			<td>99722</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot SD Semi-Dry Transfer Cell</td>
			<td>Bio-Rad Laboratories, Inc.&#160;</td>
			<td>1703940</td>
			<td>Transfer machine</td>
		</tr>
		<tr>
			<td>Transfer pipette, 3.5 mL</td>
			<td>SARSTEDT</td>
			<td>86.1171.001</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of low endotoxin content extracellular vesicles derived from cancer cell lines

细胞外囊泡（EV）是细胞在 体外 和 体内释放的膜囊泡的异质群。它们作为生物信息载体的无所不在和重要作用使它们成为有趣的研究对象，需要可靠和重复的方案来分离它们。然而，充分发挥他们的潜力是困难的，因为他们的研究仍然存在许多技术障碍（如适当的收购）。本研究提出了一种基于差异离心从肿瘤细胞系培养上清液中分离小型 EV（根据 MISEV 2018 命名法）的方案。该协议包括有关如何在分离电动汽车期间避免内毒素污染以及如何正确评估它们的指南。电动汽车的内毒素污染会严重阻碍后续实验，甚至掩盖其真正的生物效应。另一方面，被忽视的内毒素的存在可能会导致错误的结论。当提到免疫系统细胞（包括单核细胞）时，这一点尤其重要，因为单核细胞构成了对内毒素残基特别敏感的群体。因此，强烈建议筛查 EV 的内毒素污染，尤其是在处理内毒素敏感细胞（如单核细胞、巨噬细胞、髓源性抑制细胞或树突状细胞）时。

该方案的先决条件或强制性步骤是从试剂中排除可能的内毒素污染。所有使用的试剂，如FBS、DMEM、RPMI、PBS，甚至超速离心管，都必须不含内毒素（<0.005 EU/mL）。维持无内毒素污染并不容易，因为例如，用于细胞培养的常规/标准血清可能是其丰富的来源（0.364 EU/mL;见 表1）。
尽管该协议旨在分离内毒素含量低的EV，但表征分离的EV非常重要。在这项研究中，从两种...

Watch this Scientific Journal Video about Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines at JoVE.com

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

拟议的方案包括有关如何在从细胞培养上清液中分离细胞外囊泡以及如何正确评估它们期间避免内毒素污染的指南。

分离来自癌细胞系的低内毒素含量细胞外囊泡

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and ...

该协议提供了有关如何在从细胞培养上清液中分离细胞外囊泡以及如何正确评估它们期间避免内毒素污染的指南。该协议的主要优点是，每个与电动汽车有关的实验室都可以通过保持无菌程序和使用简单且广泛使用的设备来限制内毒素污染。每个第一次尝试这种技术的人都应该从低内毒素、非热原标记的新培养基、试剂和塑料器皿开始。对于该方案，请使用无内毒素试剂、水、培养基、过滤的 PBS、超低内毒素 FBS 和超速离心管。首先，将先前培养的SW480和SW620细胞系中的上清液收集到适当标记的15毫升管中。通过在室温下以500×g离心上清液5分钟来除去细胞碎片。在不干扰碎片的情况下，将上清液收集到新的标记管中，并在4摄氏度下以3，200×g离心12分钟。小心地将45毫升上清液转移到垂直放置的无菌50毫升管中，不要润湿管的边缘。用无菌透明薄膜包裹管盖，并将其垂直存放在零下80摄氏度，以便随后分离细胞外囊泡。通过制备含有约90毫升上清液的0.22微米注射器过滤器，注射器和管开始分离。用上清液填充注射器，并将过滤器固定在针头适配器上。通过过滤器将上清液过滤到50毫升管中。将七毫升滤液移液到准备好的超速离心管中，并在4摄氏度下以100， 000×g离心两小时。使用无菌巴斯德移液管丢弃上清液。使用长过滤移液器尖端将颗粒拉入超速离心管中。通过加入七毫升过滤的无内毒素PBS冲洗细胞外囊泡。超速离心后，使用无菌巴斯德移液管弃去上清液，并将沉淀重悬于200微升无内毒素PBS中。将悬浮液转移到无菌的1.5毫升低蛋白结合试管中。然后将10微升悬浮液转移到新的1.5毫升管中进行进一步分析。包裹管子的盖子，并将其存放在零下80摄氏度。现在，在新管中，使用过滤的PBS以1比1000的比例稀释一微升悬浮液，以进行纳米颗粒跟踪分析。要进行印迹分析，首先将20 μg样品与上样缓冲液混合。将样品在 70 摄氏度下孵育 10 分钟。然后用SDS将20微克样品加载到10至14%聚丙烯酰胺凝胶的每个孔中，并在150伏电压下使用电泳缓冲液进行电泳45分钟。接下来，将凝胶放入带有Towbin缓冲液的转印机中，并以25伏电压将蛋白质半干转印到聚偏二氟乙烯膜上一小时。用1%BSA在摇杆上孵育一小时来封闭膜。将BSA稀释的抗体（抗CD9或抗Alix）加入膜中，并在摇臂上以4摄氏度孵育过夜。去除抗体后，用10毫升TBST洗涤膜三次，持续10分钟。现在加入山羊抗兔或抗小鼠二抗，并在室温下在摇杆上再次孵育一小时。丢弃抗体，并如前所述清洗膜。将一毫升含有底物和鲁米诺的溶液以相等的比例倒在膜上。立即将膜置于成像系统中，并在屏幕上可视化蛋白质条带。在本研究中，蛋白质印迹分析证实了两种细胞外囊泡标志物CD9和Alix的存在。从SW480和SW620分离的囊泡的平均大小和浓度相似。用不同剂量的脂多糖（LPS）刺激单核细胞，显示使白细胞介素10分泌的最低剂量LPS和单核细胞中的肿瘤坏死因子为每毫升50皮克。显色LAL测试表明，两种细胞系的细胞外囊泡的LPS污染约为每毫升50皮克。要记住的最重要的事情是使用不含LPS或耗尽的试剂，并在程序的每一步进行常规LPS测量。防止内毒素污染比消除内毒素更容易。拟议的方案限制了由于内毒素污染EV而导致错误结果的可能性，并允许评估每个细胞的EV活性。所提出的方案对于处理内毒素敏感细胞（如单核细胞、树突状细胞和巨噬细胞）的研究人员很有用。

Research

JoVE Journal

Immunology and Infection

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

胸腺淋巴瘤的T细胞系的推导 ATM - / -</sup P53 - / -</sup鼠标

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

科研

免疫与感染

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Epstein - Barr病毒生长转化的淋巴细胞系的建立

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

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 ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

流量Virometry来分析病毒颗粒的抗原谱和外囊泡

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

成年日本血吸虫细胞外囊泡的分离与鉴定

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

利用超滤离心技术分离小鼠支气管肺泡灌洗液中的细胞外囊

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

高产细胞外囊泡制剂的纯化远离病毒

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

铁限分枝杆菌产生的细胞外囊泡的分离和特征

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...