我们感谢保科沃斯和埃夫罗斯瓦西利乌博士实验室的协助，并提供小鼠巨噬细胞，分别。我们也感谢我们的实验室经理Richard Criasia与材料的指导和帮助。最后，作者感谢Ramanpreet，考尔的帮助视频制作。

1。组织文化<ol><li>使用小家鼠巨噬细胞（BALB / c小鼠）对霍乱毒素的测定。最初文化M.肌细胞按照厂商的说明。 </li><li>传播的BALB / c小鼠细胞的Dulbecco改性鹰的中等与L-谷氨酰胺10％胎牛血清完成，并用10％胎牛血清，5％大号-谷氨酰胺和1完成1％抗生素/抗真菌剂或RPMI 1640基地媒体％抗生素/抗真菌试剂。 </li><li>培养细胞在75 厘米康宁烧瓶，在37℃，95％空气和5％CO 2通气帽。 </li><li>人类肠道上皮细胞在24小时内交货测定脂肪酸测定按照生产商的说明。 </li><li>在75 厘米康宁烧瓶通风上限在HybriCare媒体在37℃下，用10％FBS和30 ng / ml的人EGF完成人类的肠上皮细胞传播不要使用抗生素/抗真菌试剂生产商的指示。 </li><li>斯普利特所有细胞（1:3）约70％汇合。 </li><li>冻结，并把整个研究过程中使用标准的冻结协议的所有细胞。 </li><li> 注：对于较大规模的脂肪酸浓度测定，使用更快的成长，更容易维护，最初小鼠巨噬细胞。对于判罚尺度的脂肪酸浓度测定，使用人体上皮细胞。使用小鼠巨噬细胞的所有霍乱毒素治疗（见讨论）。 </li></ol> 2。脂肪酸和霍乱毒素治疗<ol><li>油酸，亚油酸和亚麻酸的转移，从公司提供的玻璃安瓿消毒的玻璃小瓶。 </li><li>每个脂肪酸转移到一个单独灭菌的Eppendorf管中，先溶解在100％乙醇为1:6的稀释，然后在终浓度为10微克/微升的RPMI 1640不完全介质。 </li><li>涡每个解决方案和互感器呃，用于存储在冷冻机（-20℃）的玻璃小瓶中。 </li><li>板细胞以2500细胞/孔在96孔组织培养板中。 </li><li>添加适当的完整的媒体带来的总孔体积为200μl准备MTT法检测细胞脂肪酸治疗。 </li><li>经过24小时的增殖期，取出纸张，更换新鲜的培养基。 </li><li>添加合适浓度的井进行测试，以每个脂肪酸（见结果）。将完整的媒体带来的以及总体积为200μl。 </li><li>孵育前24小时开始MTT法处理板。 </li><li>溶解于PBS中的浓度为1毫克/毫升和等分试样的溶液，在离心管中的霍乱毒素和存储的离心管中，在2-8℃。 </li><li>对于细胞治疗，在任一灭菌的PBS（pH 7.4）中，或者不完整，DMEM培养液1:100稀释的毒素作最后的工作浓度为1 ng /μl的。 </li><li>霍乱毒素处理细胞MTT法，如上述的板的细胞制备。 </li><li>经过24小时的增殖期，加井脂肪酸的应用（上）使用的程序进行测试，以适当的浓度霍乱毒素（浓度，看到的结果）。 </li><li>孵育前24小时开始MTT法处理板。 </li><li>整个文件作为一个正（正C）和阴性对照（阴性），为脂肪酸和霍乱毒素处理，与完全培养基和70％的乙醇，分别孵育。 </li><li>对于所有的样品和治疗，至少使用3次重复组（n = 3），阳性对照组（n = 6在这项研究中）复制。 </li></ol> 3。 MTT法<ol><li>做一个的MTT原液的浓度为2.5毫克/毫升。 </li><li>将上述溶液在96孔板的每个孔中进行测试，用200μl新鲜的完全培养基溶液取代。 </li><li>广告≤10微升MTT溶液，以每孔。 </li><li>孵育板在37℃3-4小时。 </li><li>丢弃媒体解决方案，向每孔添加100μl的1M HCl的异丙醇0.04。 </li><li>将培养板在室温下五分钟。 </li><li>从各孔将该溶液转移到一个新的离心管中。 </li><li> ，在室温下，以20,000×g离心1分钟，或直至颗粒形成。 </li><li>将20-40微升每个样品之​​间酶标仪。 </li><li>用分光光度计在570 nm处的吸光度。 </li></ol>

<ol>
	<li>Franceur, M. L., Golden, G. M., Potts, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oleic+Acid:+Its+effects+on+stratum+corneum+in+relation+to+(trans)+dermal+drug+delivery.">Oleic Acid: Its effects on stratum corneum in relation to (trans) dermal drug delivery.</a> Pharm Res. 7 (6), 621-627 (1990).</li><li>Tandon, 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=X-ray+diffraction+and+spectroscopic+studies+of+oleic+acid-sodium+acetate.">X-ray diffraction and spectroscopic studies of oleic acid-sodium acetate.</a> Chem. Phys. Lipids. 109 (1), 37-45 (1990).</li><li>Kris-Etherton, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+debate+about+n-6+polyunsaturated+fatty+acid+recommendations+for+cardiovascular+health.">The debate about n-6 polyunsaturated fatty acid recommendations for cardiovascular health.</a> Journal of the American Diabetic Association. 110 (2), 201-204 (2010).</li><li>Rizzo, W. B., Phillips, M. 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=Adrenoleukodystrophy:+dietary+oleic+acid+lowers+hexacosanoate+levels.">Adrenoleukodystrophy: dietary oleic acid lowers hexacosanoate levels.</a> Annals of Neurology. 21 (3), 232-239 (1987).</li><li>Bozan, B., Temelli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+composition+and+oxidative+stability+of+flax,+safflower+and+poppy+seed+and+seed+oil.">Chemical composition and oxidative stability of flax, safflower and poppy seed and seed oil.</a> Bioresource Tech. 99, 6354-6359 (2005).</li><li>Krein, S., Meldurm, H., Hawkins, S., Foy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+benefits+of+conjugated+linoleic+acid+to+3-dimensional+wrinkle+morphology.">Clinical benefits of conjugated linoleic acid to 3-dimensional wrinkle morphology.</a> J. American Academy of Dermatology. 60 (3), AB30 (2009).</li><li>Yu, Y., Correll, P. H., Heuvel, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugated+linoleic+acid+decreases+production+of+pro-inflammatory+products+in+macrophages:+Evidence+for+a+PPARy+dependent+mechanism.">Conjugated linoleic acid decreases production of pro-inflammatory products in macrophages: Evidence for a PPARy dependent mechanism.</a> Biochimica et Biophysica Acta. 1581 (3), 89-99 (2002).</li><li>Palombo, J., Ganguly, A., Bistrian, B., Menard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anti-proliferative+effects+of+biologically+active+isomers+of+conjugated+linoleic+acid+on+human+colorectal+and+prostatic+cancer+cells.">The anti-proliferative effects of biologically active isomers of conjugated linoleic acid on human colorectal and prostatic cancer cells.</a> Cancer Letters. 177, 163-172 (2002).</li><li>Granda, M., Sinclair, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acids+and+obesity.">Fatty acids and obesity.</a> Current Pharm. Design. 15 (36), 4117-4125 (2009).</li><li>Rosenstein, E., Kushner, L., Kramer, N., Kazandjian, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pilot+study+of+dietary+fatty+acid+supplementation+in+the+treatment+of+adult+periodontitis.">Pilot study of dietary fatty acid supplementation in the treatment of adult periodontitis.</a> Prostaglandins, Leukotrienes and Essential F.A. 68 (3), 213-218 (2003).</li><li>Ferretti, A., Flanagan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antithromboxane+activity+of+dietary+alpha-linolenic+acid:+a+pilot+study.">Antithromboxane activity of dietary alpha-linolenic acid: a pilot study.</a> Prostaglandins Leukot. Essent. Fatty Acids. 54 (6), 451-455 (1995).</li><li>Arpita, C., Pradeep, K. D., Chowdhury, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Fatty+Acids+and+Cholesterol+Present+in+Bile+on+Expression+of+Virulence+Factors+and+Motility+of+Vibrio+cholera.">Effect of Fatty Acids and Cholesterol Present in Bile on Expression of Virulence Factors and Motility of Vibrio cholera.</a> Infection and Immunity. 75 (4), 1946-1953 (2007).</li><li>Yamasaki, S., Asakura, M., Neogi, S. B., Hinenoya, A., Iwaoka, E., Aoki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+virulence+potential+of+Vibrio+cholerae+by+natural+compounds.">Inhibition of virulence potential of Vibrio cholerae by natural compounds.</a> Indian J. Med. Res. 133 (2), 232-239 (2011).</li><li>Hendriksen, R. S., Price, L. B., 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=Population+Genetics+of+Vibrio+cholerae+from+Nepal+in+2010:+Evidence+on+the+origin+of+the+Haitian+outbreak.">Population Genetics of Vibrio cholerae from Nepal in 2010: Evidence on the origin of the Haitian outbreak.</a> mBio. 2 (4), 1-6 (2011).</li><li>Schaeffler, A., Gross, 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=Fatty+acid-induced+induction+of+Toll-like+receptor-4/nuclear+factor-kappa+B+pathway+in+adipocytes+links+nutritional+signaling+with+innate+immunity.">Fatty acid-induced induction of Toll-like receptor-4/nuclear factor-kappa B pathway in adipocytes links nutritional signaling with innate immunity.</a> Immunology. 126 (2), 233-245 (2009).</li></ol>

从这项研究结果，这项研究的作者没有任何金融利益。肯恩大学为这项研究提供资金FT，FT在国王郡社区学院是目前雇员。

建议浓度脂肪酸和霍乱毒素虽然脂肪酸如何增强粘膜免疫的确切机制不明，一些研究试图探讨其有益的影响。本研究的目的是提供方法来确定细胞的脂肪酸，可以容忍，以及霍乱毒素的最大浓度没有显着影响细胞的存活细胞可以容忍的最大浓度。 为了测定浓度的三脂肪酸（油酸，亚油酸和亚麻酸），以及霍乱毒素用于治疗的细胞没有显着的不利影响，对细?...

脂肪酸，如油酸，亚油酸和亚麻酸对健康的好处已经并继续记录。例如，油酸有助于渗透的亲脂性药物在体内1,2冠状动脉心脏病，降低了24％时，饱和脂肪酸3取代的，并且是用于治疗代谢性疾病如肾上腺脑白质营养不良4是X-连锁的脂肪酸代谢的遗传性疾病。一个必要的前体在哺乳动物中花生四烯酸，亚油酸（不像油酸）时由体内合成，必须通过外部信息源，例如由亚麻籽消耗。5研究表明一些有益的健康效应如亚油酸：对皮肤的抗老化性能;抗发炎的特性;降低大肠癌和前列腺癌细胞增殖;和能力对抗肥胖和推广Øf心血管健康。9 ​​亚麻酸发挥了作用，减少牙周发炎，10和调节血栓素和前列环素的生物合成。 12阿尔皮塔（Arpita）研究胆汁脂肪酸和胆固醇的影响的五霍乱弧菌的毒力因子的表达和活力。山崎13日表示，红辣椒，以及其他自然提取的化合物，甲醇提取物有可能降低生产霍乱毒素。可以想象的是考虑使用在上面的脂肪酸（如亚麻种子）通过粘膜免疫的传染病，如霍乱的预防和减轻丰富的食品。我们进行了调查，以溶解脂肪酸和确定，使用细胞增殖实验，最高浓度与人肠上皮细胞中的脂肪酸，可以承受的，没有不利影响策LL可行性。我们假设，油酸，亚油酸和亚麻酸，在较低浓度对细胞活力提供了有益的效果，但是，在较高浓度时，它们将是对细胞有毒。而且，我们溶解的霍乱毒素和霍乱毒素B亚单位的确定的最大浓度，BALB / C小鼠巨噬细胞的细胞存活率没有显着下降，可以容忍。我们推测对细胞活性的霍乱毒素的毒性作用，即使在非常低的水平。增溶霍乱毒素的方法，用它来确定的毒素，细胞没有显着下降的生存能力可以容忍的最大金额为今后的研究提供了一个优点。例如，可以使用上述的方法的组合，以确定是否脂肪酸提供抗霍乱感染的粘膜免疫的细胞。据我们所知，这种理性和方法尚未探索。 我们二scuss我们的初步数据如何可以用在以后的调查，以确定是否油酸，亚油酸和亚麻酸提供细胞粘膜抗感染霍乱。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cells/Reagent</td>
		 </tr>
		 <tr>
			<td>Mus musculus macrophages</td>
			<td>ATCC</td>
			<td>ATCC RAW 264.7</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dulbecco's Modified Eagle's Medium</td>
			<td>ATCC</td>
			<td>30-2002</td>
			<td></td>
		 </tr>
		 <tr>
			<td>L-glutamine</td>
			<td>ATCC</td>
			<td>30-2115</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal bovine serum </td>
			<td>Bio-west</td>
			<td>S0250</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Antibiotic/antimycotic </td>
			<td>Hyclone</td>
			<td>SV3007901</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Human intestinal epithelial cells </td>
			<td>ATCC</td>
			<td>ATTC CCL-241</td>
			<td></td>
		 </tr>
		 <tr>
			<td>HybriCare media </td>
			<td>ATCC</td>
			<td>46-X</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Oleic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>O1008</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Linoleic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>L-1376</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Linolenic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>L-2376</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cholera toxin</td>
			<td>Sigma-Aldrich</td>
			<td>C8052</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>BD Falcon 96-Well Cell Culture Plates</td>
			<td>BD Biosciences </td>
			<td>351172</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Spectrophotometer with Dynex Revelations 4.22 software</td>
			<td>Dynex</td>
			<td>91000101</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

determination of tolerable fatty acids and cholera toxin concentrations using human intestinal epithelial cells and balb/c mouse macrophages

脂肪酸在预防和减轻人类和非人类疾病的积极作用已经并将继续被广泛记载。这些角色包括感染性和非感染性疾病，包括预防炎症和传染病的粘膜免疫的影响。霍乱是由霍乱弧菌引起的急性肠道疾病。它发生在发展中国家，如果不及时治疗，可导致死亡。虽然霍乱疫苗存在时，它们并不总是有效的，需要其他的预防方法。我们容许浓度的三脂肪酸（油酸，亚油酸和亚麻酸）和霍乱毒素，分别使用小鼠BALB / C小鼠巨噬细胞和人类肠道上皮细胞，以确定。溶解上述脂肪酸和使用细胞增殖实验，以确定的浓度范围和特定浓度的脂肪酸，没有吨有损于人类肠道上皮细胞的存活率。增溶的霍乱毒素B亚单位，在一个试验中，并用它来确定的浓度范围和霍乱毒素，在统计学上不降低细胞存活率在BALB / C小鼠的巨噬细胞中的特定浓度的。 我们发现最佳的脂肪酸浓度介于1-5纳克/微升，和霍乱毒素&lt;30纳克每处理的。这些数据可能有助于未来的研究，旨在找到脂肪酸在预防或减轻感染霍乱的保护黏膜的作用。

脂肪酸的最佳浓度测定脂肪酸的最适浓度被定义为细胞生长的最大浓度可媲美或超过对照组细胞，在结果中的相对低的变异。 ，以确定最佳浓度的油酸，亚油酸和亚麻酸的细胞最初以较大的增量，并与不同浓度的各脂肪酸购买较小的增量处理， 图1示出的生存的细胞作为阳性对照的治疗使用的函数描绘脂肪酸浓度的增加。显而易见的是，细胞不耐受超过100毫微克/微...

Watch this Scientific Journal Video about Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages at JoVE.com

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

我们容许浓度的三脂肪酸（油酸，亚油酸和亚麻酸）和霍乱毒素没有显着不利影响细胞生存脂肪酸和毒素溶解，并利用他们在细胞存活实验来确定。

使用人类肠道上皮细胞和BALB / C小鼠巨噬细胞的容忍脂肪酸和霍乱毒素浓度的测定

The positive role of fatty acids in the prevention and alleviation of non-human and  human diseases have been and continue to be extensively documented. ...

determination-tolerable-fatty-acids-cholera-toxin-concentrations

Research

JoVE Journal

Immunology and Infection

9.9K 次观看.  Kingsborough Community College. 我们容许浓度的三脂肪酸（油酸，亚油酸和亚麻酸）和霍乱毒素没有显着不利影响细胞生存脂肪酸和毒素溶解，并利用他们在细胞存活实验来确定。

视频: Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

科研

免疫与感染

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

肠道细菌侵入肠上皮细胞<em&gt;体外</em&gt;使用垂直扩散室模型，显着增强

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

哺乳动物细胞细菌挑战后应激颗粒形成的免疫荧光分析

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated fatty acids, which might play a critical role in regulating their immune functions. Numerous studies have shown that various fatty acids, saturated or unsaturated, may possess different impacts on cell growth and function. However, the approaches used for fatty acid preparation vary, which may lead to non-physiological results. Serum albumin, a natural carrier for fatty acids in mammalian peripheral blood, is recommended for forming a conjugate complex with the sodium salt of fatty acids to study fatty acid function in mammalian cells, thus minimizing the toxicity of fatty acid soap. Thus, a simple, relatively quick heating and sonicating method is developed and presented here for BSA-fatty acid conjugate formation. We describe a protocol using saturated fatty acids, especially stearic acids to induce severe cell death in mouse bone-marrow derived macrophages. We further demonstrate that saturated fatty acid-induced cell death is positively associated with accumulated cellular ceramide levels. This method can be extended for studies of the impact of fatty acid on other mammalian cells.

We illustrate a straight-forward method to derive murine primary macrophages from bone marrow cells and a simple method to prepare BSA-fatty acid conjugates. Then we demonstrate that saturated fatty acids can induce macrophage cell death, and such cell death is positively associated with cellular accumulation of ceramide levels.

饱和脂肪酸诱导神经酰胺相关巨噬细胞死亡

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

小鼠和人源性胃上皮单层培养对再生的研究

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

诱导BALB/c小鼠药物诱导的自身免疫性肝炎研究其致病机制

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

利用人类诱导的多能干细胞衍生的肠道有机体研究和改性上皮细胞对沙门氏菌和其他病原体的保护

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...