谢谢艾琳娜Mitsi（研究助理）和Sr阿玲汉考克（研究护士）和剧院工作人员对拍摄的贡献，并以邵大卫（研究护士），他与我们的研究支气管镜的支持。还要感谢我们的志愿者丽贝卡·邓菲允许此过程被拍摄下来。

1。本主题由呼吸研究护士在飞抵<ol><li>这个问题已经被禁食&gt; 4小时固体食物和2小时为透明液体1。 </li><li>该过程执行了一天的情况下用适当的预处理和后处理过程检查（按照标准的医院政策）。 </li><li>主题穿着医院的病号服。 </li><li>一个小规套管插入（理想为左手），并保持在原位 ，直至后程序恢复期间1的端部。 </li><li>操作前观察被当作（血压，心脏率，血氧饱和度[SATS]）。 </li><li>受试者被转移到支气管镜套装。 </li></ol> 2。本主题由呼吸研究临床医生在经历了支气管镜检查准备在套件的程序<ol><li>监控设备连接，其中包括：3导联心电图，脉搏血氧仪1,10和血压计。 Anesthet不需要IC支持，包括书面知情同意书。 </li><li>氧气可以通过鼻导管高达4升/分钟交付，实现SATS&gt; 90％1。 </li><li>操作员检查支气管镜，这包括通过抽吸无菌生理盐水确保有效的吸力。 </li><li>局部麻醉用利多卡因在鼻通道（使用Instillagel 1）和口腔粘膜实现（使用利多卡因） </li><li>如果镇静是适当的，由主体要求，咪达唑仑静脉注射给药1。适当的逆转剂（氟马西尼）是立即可用。 </li><li>复苏设备应随时可用1,10。 </li></ol> 3。支气管镜插入并定位<ol><li>插管通过鼻子通常是。如果这是不可能的，由于鼻息肉，鼻甲发炎，或任何不适那么受试者经口插管（口后卫是用来防止损坏的范围或受试者的牙齿）。 </li><li>局部麻醉在喉是使用4％利多卡因完成，通常总共4-6毫升被使用。这通常导致咳嗽的灌输。 </li><li>声带传递，并用2毫升分装2％利多卡因在隆突，在右下肺叶（RLL）及右肺中叶（RML）的划分，并在入口处RML进一步黏膜麻醉。 </li><li>支气管镜定位在RML内，最好在内侧段，在一个位置，它足以在一个安全的位置的远端，但不能太远侧使气道塌陷，当施加抽吸（&#39;表情试验“使用抽吸按钮）。 </li><li>良好的定位是，可以通过支气管镜和不完全关闭紧随温和的吸力气道被完全保持在一个位置支气管镜眨眼测试期间表示。 </li></ol> 4。 BAL中进行<ol><li>四60毫升注射器预填充有温热生理盐水 - 60毫升，50毫升，50毫升，40毫升连续注射器。当主体和支气管镜准备好生理盐水的第一注射器是由支气管镜辅助灌输，而支气管镜保持在RML的位置。 </li><li>然后，通过使用相同的端口和50 ml注射器辅助进行温柔的手吸。 </li><li>这个过程，然后再重复3次，用在我们的具体技术中使用200毫升的最大体积。 </li><li>检索BAL液轻轻挤入已经举行了融冰标签的容器。玻璃容器可以presiliconized以抑制细胞附着和细胞最大化回报。 </li><li> BAL液出现朦胧的反对与表面活性剂形成表面的肥皂泡的光。 </li><li>支气管镜慢慢地完全撤出。 </li><li> BAL液被输送无延迟冰上立即处理。 </li></ol> 5。待恢复</p&gt; <ol><li>这个问题是由一个病房的呼吸研究护士恢复2-4小时。 </li><li>在此期间，他们被允许休息，然后只吃喝&gt; 60分钟1手术后（当他们的燕子被评定为安全），为了减少误吸的危险。 </li><li>发表意见的程序进行监控和记录。临床检查时在出院前。 </li><li>这个题目是介绍出院前对常见的副作用（发热，轻度右submammary胸部不适，咽 ​​痛），并给出显著副作用发生1的情况下的联系人号码。 </li><li>这个题目是跟进在1-5天之后程序与临床联系。这可能是通过电话或亲自。 </li></ol> 6。细胞被隔离在实验室 - 执行对冰川消融，在可能的情况<ol><li> BAL液的体积是通过无菌纱布拭子双层记录和过滤去除黏液栓到预冷，无菌50ml离心管中。 </li><li>细胞通过离心分离，在500×g离心沉淀，在4℃下进行5-10分钟，并通过涡旋在50毫升冷生理盐水洗涤。离心分离重复一次。 </li><li>细胞再悬浮于培养基（RPMI-1640 +10％胎牛血清+ 2 2mM L-谷氨酰胺+青霉素[40国际单位/毫升]，链霉素[75国际单位/毫升]，和两性霉素B [0.5国际单位/毫升]）和使用台盼蓝等体积和血细胞计数器计数的差分进行。 </li><li>细胞悬浮液被归一化到所要求的密度（我们使用1×10 6个细胞/ ml），通过移液导入适当组织培养板中，并在37℃下进行3小时，使巨噬细胞的粘附。 1毫升每孔该细胞悬浮液适用于24孔培养板中。 </li><li>对于淋巴细胞工作，3小时温育后，将培养基用移液管轻轻地上下3倍，并收集。此培养基中含有淋巴细胞和非粘附的巨噬细胞，并且可以用来评估淋巴细胞的功能，它也可在纯化步骤中使用，例如与CD14磁珠除去巨噬细胞。 </li><li>对巨噬细胞的工作，3小时温育后，将培养基小心地用最少的移液除去，用不含抗生素的新鲜，温暖培养基（RPMI 1640 +10％胎牛血清+ 2 2mM L-谷氨酰胺）取代。巨噬细胞是粘附于组织培养板中（参见图1）。 </li></ol>

<ol>
	<li>BTS, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+British+Thoracic+Society+Bronchoscopy+Guideline+Committee+-+A+sub-Committee+of+the+Standards+of+Care+Committee+of+the+British+Thoracic+Society.">The British Thoracic Society Bronchoscopy Guideline Committee - A sub-Committee of the Standards of Care Committee of the British Thoracic Society.</a> Thorax. 56 (Suppl I), 1-21 (2001).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+recommendations+and+guidelines+for+bronchoalveolar+lavage+(BAL)+report+of+the+European+Society+for+Pneumology+Task+Group.">Technical recommendations and guidelines for bronchoalveolar lavage (BAL) report of the European Society for Pneumology Task Group.</a> Eur. Respir. , 561-585 (1989).</li><li>Gordon, S. B., Irving, G. R., Lawson, R. A., Lee, M. E., Read, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+trafficking+and+killing+of+Streptococcus+pneumoniae+by+human+alveolar+macrophages+are+influenced+by+opsonins.">Intracellular trafficking and killing of Streptococcus pneumoniae by human alveolar macrophages are influenced by opsonins.</a> Infect. Immun. 68, 2286-2293 (2000).</li><li>Gordon, S. 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=Pulmonary+immunoglobulin+responses+to+Streptococcus+pneumoniae+are+altered+but+not+reduced+in+human+immunodeficiency+virus-infected+Malawian+adults.">Pulmonary immunoglobulin responses to Streptococcus pneumoniae are altered but not reduced in human immunodeficiency virus-infected Malawian adults.</a> J. Infect. Dis. 188, 666-670 (2003).</li><li>Fullerton, D. 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=Domestic+smoke+exposure+is+associated+with+alveolar+macrophage+particulate+load.">Domestic smoke exposure is associated with alveolar macrophage particulate load.</a> Trop. Med. Int. Health. 14, 349-354 (2009).</li><li>Jambo, K. 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=Bronchoalveolar+CD4++T+cell+responses+to+respiratory+antigens+are+impaired+in+HIV-infected+adults.">Bronchoalveolar CD4+ T cell responses to respiratory antigens are impaired in HIV-infected adults.</a> Thorax. 66, 375-382 (2011).</li><li>Eagan, 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=Lung+fluid+immunoglobulin+from+HIV-infected+subjects+has+impaired+opsonic+function+against+pneumococci.">Lung fluid immunoglobulin from HIV-infected subjects has impaired opsonic function against pneumococci.</a> Clin. Infect. Dis. 44, 1632-1638 (2007).</li><li>Rose, A. S., Knox, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchoalveolar+lavage+as+a+research+tool.">Bronchoalveolar lavage as a research tool.</a> Sem. Respir. Crit. Care Med. 28, 561-573 (2007).</li><li>Wright, A. K. 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=Human+Nasal+Challenge+with+Streptococcus+pneumoniae+is+Immunising+in+the+Absence+of+Carriage.">Human Nasal Challenge with Streptococcus pneumoniae is Immunising in the Absence of Carriage.</a> PLoS Pathog. , (2012).</li><li>Bolliger, C. 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=ERS/ATS+statement+on+interventional+pulmonology+European+Respiratory+Society/American+Thoracic+Society.">ERS/ATS statement on interventional pulmonology European Respiratory Society/American Thoracic Society.</a> Eur. Respir. J. 19, 356-373 (2002).</li><li>Ettensohn, D. B., Jankowski, M. J., Duncan, P. G., Lalor, 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=Bronchoalveolar+lavage+in+the+normal+volunteer+subject.+I.+Technical+aspects+and+intersubject+variability.">Bronchoalveolar lavage in the normal volunteer subject. I. Technical aspects and intersubject variability.</a> Chest. 94, 275-280 (1988).</li><li>Ettensohn, D. B., Jankowski, M. J., Redondo, A. A., Duncan, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchoalveolar+lavage+in+the+normal+volunteer+subject.+2.+Safety+and+results+of+repeated+BAL,+and+use+in+the+assessment+of+intrasubject+variability.">Bronchoalveolar lavage in the normal volunteer subject. 2. Safety and results of repeated BAL, and use in the assessment of intrasubject variability.</a> Chest. 94, 281-285 (1988).</li><li>De Brauwer, E. I., Jacobs, J. A., Nieman, F., Bruggeman, C. A., Drent, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchoalveolar+lavage+fluid+differential+cell+count.+How+many+cells+should+be+counted.">Bronchoalveolar lavage fluid differential cell count. How many cells should be counted.</a> Anal. Quant. Cytol. Histol. 24, 337-341 (2002).</li><li>Mtunthama, 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=Malawians+permit+research+bronchoscopy+due+to+perceived+need+for+healthcare.">Malawians permit research bronchoscopy due to perceived need for healthcare.</a> J. Med. Ethics. 34, 303-307 (2008).</li><li>Pereira, W., Kovnat, D. M., Snider, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prospective+cooperative+study+of+complications+following+flexible+fiberoptic+bronchoscopy.">A prospective cooperative study of complications following flexible fiberoptic bronchoscopy.</a> Chest. 73, 813-816 (1978).</li><li>Huang, Y. C., Bassett, M. A., Levin, D., Montilla, T., Ghio, 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=Acute+phase+reaction+in+healthy+volunteers+after+bronchoscopy+with+lavage.">Acute phase reaction in healthy volunteers after bronchoscopy with lavage.</a> Chest. 129, 1565-1569 (2006).</li><li>Gritzfeld, J. 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=Experimental+Human+Pneumococcal+Carriage.">Experimental Human Pneumococcal Carriage.</a> J. Vis. Exp. (72), (2013).</li></ol>

安德烈M.柯林斯是由比尔和梅林达·盖茨基金会，也没有潜在的利益冲突提供资金。她收到的资金从默克（MSD）和葛兰素史克公司出席会议和讲课费。 50研究BAL中独立进行的。 杰米Rylance由威康信托基金资助，并具有无利益冲突。他没有收到任何其他资金。 70研究BAL中独立进行的。 丹尼尔G.伍顿是由NIHR博士研究生奖学金资助，并具有无利益冲突。 40研究BAL中独立进行的。 安吉拉·莱特有没有潜在的利益冲突。协助超过100个研究BAL中。 亚当KA Wright有没有潜在的利益冲突。他已经处理了超过50 BAL样本。 邓肯G.富勒顿 </strong&gt;有没有潜在的利益冲突。 100研究BAL中独立进行的。 斯蒂芬B.戈登有没有潜在的利益冲突。他曾获得的资金从默克（MSD）和诺华出席会议。 400研究BAL中独立进行的。

为了达到良好的效果，以下是有所帮助： <ol><li>好anesthetisation在舌和后口咽部的基极。这很重要，因为它提高了主体的程序宽容和整体舒适度。 </li><li>无论是儿科（外径为3.4-3.6毫米）或成年支气管镜（外径为4.8-5.9毫米）都可以使用。 </li><li>如果有一个在鼻腔气管插管任何不适，口腔插管是首选。一般来说鼻腔插管可以是一个简单的技术或应该放弃。 </li><li>利多卡因对口咽初始滴注后，受试者可能更喜欢支气管镜被撤回以待麻醉发作，尤其是如果支气管镜经口插入。 </li><li>确保至少有10×10毫升注射器预填充只有2-4毫升的利多卡因。空气应留在注射器使麻醉的改进提供直接到线和支气管黏膜SA。 </li><li>因为在操作过程中咳嗽显著降低BAL产率;用温热的盐水，并具有良好的粘膜麻醉是重要的，以减少此。 </li><li> 50ml的注射器必须预先填充用温盐水。这是常见的前50 ml生理盐水滴注，以产生由于死空间损耗和运行到终端呼吸道其余的10-20毫升。进一步等份返回流体的比例更大。 </li><li>操作人员应经常检查支气管镜前插管，包括确保有效吸吸用无菌生理盐水。吸力的损失可由于管或周围的支气管镜或抽吸装置的连接泄漏的扭结普遍发生。 </li><li>在手动吸，最好呼吸道应该不会崩溃。如果吸入循环地折叠该气道，BAL回报减少到一个阀机构，和局部炎症和出血的可能性更大：这反过来又使呼吸道米矿石烦躁和咳嗽的可能性较大。选择最合适的RML亚段分支，并使用低吸气压力，因此，这两个重要的。 </li><li>当空气被不断吸入重新检查注射器向气管镜的连接以及在所述远端气道的支气管镜的位置。如果这是一个经常出现的问题，后期的程序保证有在支气管镜本身没有漏洞。 </li><li> BAL从吸烟者往往较深由于细胞内颗粒的存在。吸烟者可能容忍支气管镜较差，并有咳嗽加重。 </li><li>优选的BAL量产量&gt; 100毫升。为&gt; 150 ml具有产量非常出色。我们没有经历过任何产量&gt; 170毫升还有就是细胞产量和BAL量产量之间没有线性关系。 </li><li>的&lt;100毫升收益率，根据我们的经验，更可能导致副作用，如咳嗽，胸痛和发烧。我们建议受试者&lt;100毫升的产量是RECO维雷德在左侧卧位，这样可以帮助任何剩余肺液引流。 </li><li>临床接触应使与主体在1-5天，以确保没有副作用。 </li><li>咪达唑仑镇静用于&lt;50％的受试者在英国和&lt;5％，在马拉维。蓝精灵似乎并没有直接提高耐受性，一些经营者认为，镇静有时可能适得其反维持与主题一致的（和令人放心的）对话。 </li><li>根据我们的经验，有这种技术一个显著的学习曲线。例如医生和护士之间的协调需要时间和收益率往往较低在学习过程的开始。 </li><li>在HIV阳性者中，我们使用抗生素预防prebronchoscopy。 </li></ol>

背景纤维支气管镜在临床实践中形成的，它被广泛用于两个及诊断治疗1,2。支气管肺泡灌洗（BAL），去除未贴壁细胞，并从粘膜表面肺衬液，活检用于采样的粘膜和粘膜下组织的。在研究环境，BAL使肺部内采样的先天（肺巨噬细胞3-5），细胞（B-和T-细胞6）和体液（免疫球蛋白7）的反应。 BAL是国际公认的用于研究目的8，自1999年该技术已在&gt; 1000例受试者完成了在英国和马拉维由本集团。我们使用这种技术的肺炎球菌抗原与生俱来的，细胞和体液免疫反应，包括人体实验肺炎球菌马车9,17，生物质烟雾5，艾滋病毒和疫苗接种，并辅助治疗STU研究死成肺炎恢复。我们的技术使用滴注液轻柔手持式抽吸，这样的设计最大限度地返回BAL量和应用上的纤毛上皮细胞的最小剪切力，以BAL液中保存细胞的结构和功能。 在研究方面BAL利用从呼吸和重症监护医师（通常被称为支气管冲洗，洗涤，冲洗，或BAL）谁的目标是要获得诊断或治疗的好处实行不同的技术。研究方法被设计为收获细胞，并保持生存力，以促进细胞在体外培养中的生长。由于这些原因，研究技术使用较大体积的滴注液（通常在200毫升的数量级），并采用手动吸减少细胞损伤。患者给予局部麻醉剂，提供清醒镇静（咪达唑仑），并以最小的副作用耐受的过程良好。口头和书面的主观科技信息提高了宽容和书面知情同意书是强制1。 目标总的目标是，程序应该是安全和有效的。主题应该不会遇到任何生理干扰和运营商应该坚持收集超过从受试者百毫升BAL的。手术后，受试者应该经历最小的副作用。 安全受试者的安全是至关重要的。主题使用明确的纳入和排除标准精心挑选的。该协议包括的潜在风险，以及减轻他们所采取的步骤的描述。 禁忌症研究支气管镜可表示为绝对或相对的，都包括在我们的研究协议作为排斥和纳入标准。我们的研究对象都是筛选，以确保完全健康。 绝对禁忌症包括颈椎不稳，反应迟钝缺氧，不稳定型心绞痛，出血素质，恶性心律失常。 相对禁忌症，包括那些具有增加的并发症发生率有关的病症，包括： 一般：不好合作课题1，任何显著一般的医疗问题如癫痫，以前的耐受性很差支气管镜检查，已知的不良反应，利多卡因或咪达唑仑，怀孕，营养差。 呼吸系统：，高碳酸血症，哮喘不稳定1，显著损害呼吸功能1（FEV 1 &lt;1升），肺动脉高压缺氧[空气&lt;94％饱和度（SATS）]。 心血管：尿毒症，6周心肌梗塞1， 上腔静脉阻塞的范围内。 其他：免疫抑制（我们的小组会定期执行此多项式杜热在HIV阳性者）。 见表1 - BAL与研究相关 ​​的风险。

<table border="1" cellpadding="0" cellspacing="0" style="width:966px;" width="966">
	<tbody>
		<tr>
			<td style="width:244px;height:25px;">
			Material
			</td>
			<td style="width:161px;height:25px;">
			Company
			</td>
			<td style="width:151px;height:25px;">
			Catalogue Number
			</td>
			<td style="width:410px;height:25px;">
			Comments
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Fibreoptic bronchoscope, light and suction source
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Surgical gown, sterile gloves
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Sphygnomanometer
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For continuous patient monitoring
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Pulse oximeter
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For continuous patient monitoring
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			3 lead ECG monitor
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For continuous patient monitoring
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Nasal oxygen delivery 2-4l/min
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Lidocaine 10% spray (Xylocaine)
			</td>
			<td style="width:161px;height:26px;">
			AstraZeneca
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For topical anaesthesia
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Lidocaine hydrochloride 2%
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For topical anaesthesia
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Lidocaine gel (Instillagel)
			</td>
			<td style="width:161px;height:26px;">
			Farco-Pharma, Germany
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			Alternative products available from other suppliers
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Normal saline, sterile 200ml
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			Warm to 30oC
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			10ml taper-end syringe (x10)
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For administration of lidocaine
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Intravenous cannula 18G
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For administration of midazolam
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Midazolam
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For sedation
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Flumazenil
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			Reversal of benzodiazepine sedation (emergency use only)
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			60ml taper-end syringes (x4)
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For normal saline injection and BAL fluid withdrawal.
			May require connector to attach to the injection port of the bronchoscope
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Sterile gauze swab
			</td>
			<td style="width:161px;height:26px;">
			Vernaid, UK
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			Alternative products available from other suppliers
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Sterile container for BAL fluid
			</td>
			<td style="width:161px;height:26px;">
			See text and comments
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			Siliconised glass bottles reduce macrophage adherence. Alternatively, 50ml centrifuge tubes may be used (total capacity 200ml)
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Sigmacote
			</td>
			<td style="width:161px;height:26px;">
			Sigma, UK
			</td>
			<td style="width:151px;height:26px;">
			SL2
			</td>
			<td style="width:410px;height:26px;">
			Only if siliconised glass bottles used. NB: Harmful and flammable. If used, follow precautions detailed in manufacturer&#8217;s MSDS
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Centrifuge
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;"></td>
			<td style="width:410px;height:26px;">
			At least 4x50ml tube capacity. Refrigeration to 4oC preferred.
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Shandon Cytospin centrifuge
			</td>
			<td style="width:161px;height:26px;">
			Thermo Scientific, UK
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For differential count only
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			RPMI 1640 medium with L-glutamine
			</td>
			<td style="width:161px;height:26px;">
			Sigma, UK
			</td>
			<td style="width:151px;height:26px;">
			R8758
			</td>
			<td style="width:410px;height:26px;">
			Alternative products available from other suppliers
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Fetal bovine serum
			</td>
			<td style="width:161px;height:26px;">
			Sigma, UK
			</td>
			<td style="width:151px;height:26px;">
			F6178
			</td>
			<td style="width:410px;height:26px;">
			Alternative products available from other suppliers
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Penicillin-Streptomycin
			</td>
			<td style="width:161px;height:26px;">
			Sigma, UK
			</td>
			<td style="width:151px;height:26px;">
			P4333
			</td>
			<td style="width:410px;height:26px;">
			Alternative products available from other suppliers
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Amphotericin B
			</td>
			<td style="width:161px;height:26px;">
			Sigma, UK
			</td>
			<td style="width:151px;height:26px;">
			A2942
			</td>
			<td style="width:410px;height:26px;">
			Alternative products available from other suppliers
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Tissue culture plates
			</td>
			<td style="width:161px;height:26px;">
			Greiner Bio-One, UK
			</td>
			<td style="width:151px;height:26px;">
			662160
			</td>
			<td style="width:410px;height:26px;">
			Alternative products available from other suppliers
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Glass microscope slides
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For differential count only
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Shandon cytofunnel
			</td>
			<td style="width:161px;height:26px;">
			Thermo Scientific, UK
			</td>
			<td style="width:151px;height:26px;">
			A78710003
			
			</td>
			<td style="width:410px;height:26px;">
			For differential count only
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Shandon cytoclip
			</td>
			<td style="width:161px;height:26px;">
			Thermo Scientific, UK
			</td>
			<td style="width:151px;height:26px;">
			59910052
			</td>
			<td style="width:410px;height:26px;">
			For differential count only
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Hemacolor staining set (fixative, red and blue reagents)
			</td>
			<td style="width:161px;height:26px;">
			Merck, Germany
			</td>
			<td style="width:151px;height:26px;">
			111661
			</td>
			<td style="width:410px;height:26px;">
			Use according to manufacturer&#8217;s instructions
			</td>
		</tr>
	</tbody>
</table>

bronchoalveolar lavage (bal) for research; obtaining adequate sample yield

我们描述了纤维支气管镜支气管肺泡灌洗（BAL）使用手动手持抽吸，以从粘膜表面除去非贴壁细胞和肺里流体的研究方法。在研究环境，BAL使先天（肺巨噬细胞）的采样，细胞（B-和T-细胞）和体液免疫（免疫球蛋白）肺中的反应。 BAL是国际公认的用于研究目的，自1999年该技术已在&gt; 1000例受试者完成了在英国和马拉维由本集团。 我们的技术采用滴注液的温和的手持抽吸，这是被设计来最大化BAL体积退回和应用上的纤毛上皮最小的剪切力，以维持细胞的结构和功能的BAL液中，并保持生存力，以促进生长细胞在体外培养的。因此，研究技术使用量较大插件tillate（通常在200毫升的数量级），并采用手动吸减少细胞损伤。 患者给予局部麻醉剂，提供清醒镇静（咪达唑仑），并以最小的副作用耐受的过程良好。口头和书面的主题信息提高了宽容和书面知情同意书是强制性的。受试者的安全是至关重要的。主题使用明确的纳入和排除标准精心挑选的。 该协议包括的潜在风险的描述，并减轻他们所采取的步骤，禁忌名单，前，后，过程检查，以及精确的支气管镜检查及实验室技术。

良好的临床效果都超过100毫升BAL体积和谁经历顶多一个主题，轻微不适（ 见表1）。这需要一个宽松，合作的主题。临床医生必须有信心，做好充分的准备（ 材料 ），并了解对志愿者。 在细胞产量（包括差分计数）之间和内部学科的变化进行了描述。从健康受试者细胞分类计数通常是&gt; 90％的巨噬细胞与少量的中性粒细胞（&lt;2％）和淋巴细胞（5-10％）。 BAL细胞离心到显微镜载片可以用一个差分核染色剂进行染色，以获得该信息（ 图1）。在图1中，BAL细胞已被固定，并根据制造商的说明使用Hemacolor染色染色。至少300个细胞应该算做获得可靠的结果巨噬细胞，中性粒细胞和淋巴细胞; 500个细胞的罕见的细胞类型。 <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> 风险 </td><td> 风险 </td><td> ：通过减少在这个协议 </td></tr><tr><td colspan="3"> 支气管镜和BAL 1，14 的影响 </td></tr><tr><td>轻度不适</td><td> &lt;25％14，＃ </td><td>适当的镇痛和镇静作用。 自信和关爱的方式的问题。 </td></tr><tr><td>鼻出血</td><td> &lt;1％＃ </td><td>轻度压力只有经鼻插管。 如果主体不适或缩小光圈 - 口腔插管代替。 </td></tr><tr><td>支气管出血（咯血） </td><td> &lt;0.1％* 15 </td><td>无取活检。 支气管镜检查过程中严格控制 - 避免呼吸道黏膜。 </td></tr><tr><td> Naus谈EA，呕吐及吸入性肺炎</td><td> &lt;0.02％14，＃ </td><td>禁食固体食物&gt; 4小时操作前1。 充足的局部麻醉。 过程中，半卧位。 仔细后期过程的观察。 </td></tr><tr><td>发烧</td><td> 0.01 14 - 1％＃16 </td><td>涉及炎症，并且可以通过BAL的最大集合最小化。 只有单瓣BAL。＃ </td></tr><tr><td>痛鼻/喉及声音嘶哑</td><td> &lt;25％14，＃ </td><td>鼻子，喉咙和喉部（减少咳嗽）有足够的局部麻醉。 </td></tr><tr><td>感染</td><td> &lt;在HIV阴性的0.1％， 在HIV阳性14，＃1％ </td><td>支气管标准洗涤程序1。 只有单瓣BAL。＃ 早期发现感染：在1小时内的临床检查支气管镜检查后1-5天付款交单的临床接触。 </td></tr><tr><td>胸痛/咳嗽</td><td> &lt;0.2％14，＃ </td><td>单瓣只BAL。最大BAL集合。＃ </td></tr><tr><td colspan="3"> 影响药物：利多卡因，咪达唑仑 </td></tr><tr><td>心律失常</td><td>非常罕见的* </td><td rowspan="3">最多5毫克/千克用14利多卡因。 温生理盐水注入。＃ 脉搏血氧饱和度（SATS&gt; 90％氧气补充），在整个过程心脏监测。 苯二氮卓受体拮抗剂（氟马西尼）立即可用。 </td></tr><tr><td>定向力障碍/搅拌</td><td> &lt;0.1％†＃ </td></tr><tr><td>有氧呼吸抑制</td><td>非常罕见的* </td></tr></tbody></table> *没有经历过在我们的小组中超过1,500研究程序。 †Idiosyncratic而不是剂量依赖性反应。 ＃在超过1500名研究程序本集团的经验。 表1中。 BAL与研究相关 ​​的风险 。这包括危险或风险，这是如何减少使用所描述的技术。纤支镜/ BAL，也是用任何药物药物的作用效果均包括在内。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/4345/4345fig1highres.jpg" width="500" /> 图1。显微镜幻灯片显示BAL巨噬细胞和中性粒细胞偶见 。 BAL细胞已被固定在甲醇并用Hemacolor红（曙红Y）和蓝色（天青B）。

Watch this Scientific Journal Video about Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield at JoVE.com

Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

我们描述了使用低压吸入纤维支气管镜检查和支气管肺泡研究技术。该技术被用于从肺支气管肺泡表面收获的免疫细胞。局部麻醉剂和轻度镇静（咪达唑仑）被使用。主题容忍的程序以及经验和最小的副作用。

支气管肺泡灌洗（BAL）的研究，获得适当的采样率

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove ...

bronchoalveolar-lavage-bal-for-research-obtaining-adequate-sample

Research

JoVE Journal

Medicine

Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield

73.0K 次观看.  National Institute for Health Research. 我们描述了使用低压吸入纤维支气管镜检查和支气管肺泡研究技术。该技术被用于从肺支气管肺泡表面收获的免疫细胞。局部麻醉剂和轻度镇静（咪达唑仑）被使用。主题容忍的程序以及经验和最小的副作用。 

视频: Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human diseases.

综合胸部肿瘤生成数据库 - 翻译研究的工具

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer ...

科研

医学

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

双侧颈总动脉闭塞足够的预处理刺激诱导局灶性脑缺血早期缺血耐受

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

为获取原发性卵巢癌细胞固体样品的方法

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

与人体组织转化为癌症研究工作

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome (ARDS)

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future therapies. Severe lung injury with a reproducible deterioration of pulmonary gas exchange and hemodynamics can be induced in anesthetized pigs using repeated lung lavages with warmed 0.9% saline (50 ml/kg body weight). Including standard respiratory and hemodynamic monitoring with clinically applied devices in this model allows the evaluation of novel therapeutic strategies (drugs, modern ventilators, extracorporeal membrane oxygenators, ECMO), and bridges the gap between bench and bedside. Furthermore, induction of lung injury with lung lavages does not require the injection of pathogens/endotoxins that impact on measurements of pro- and anti-inflammatory cytokines. A disadvantage of the model is the high recruitability of atelectatic lung tissue. Standardization of the model helps to avoid pitfalls, to ensure comparability between experiments, and to reduce the number of animals needed.

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome ARDS

Repeated pulmonary lavages in anesthetized pigs induce lung injury resembling major aspects of human acute respiratory distress syndrome (ARDS). For this purpose the lungs are repeatedly lavaged with 0.9% saline at 37 &#176;C. The goal of the protocol is a reproducible mitigation of gas exchange and hemodynamics for research in ARDS.

灌洗诱发的表面活性剂耗尽猪一样的急性呼吸窘迫综合征模型（ARDS）

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future ...

Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ function. Clarifying the mechanism of blood vessel formation is therefore extremely useful for elucidating developmental processes and pathological mechanisms. Murine hind-limb vessels are often used as a model for physiological and pathological angiogenesis. Evaluation is mainly performed via a two-dimensional method using tissue sections. However, methods for evaluating three-dimensional (3D) vascular morphology are particularly limited. This paper introduces a method for visualizing murine hind-limbs using computed tomography (CT). Radiation-opaque resin is injected through the descending aorta, and whole vessels are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray CT device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels in the lower extremities. Furthermore, this method can be used to visualize all blood vessels below the diaphragm and evaluate blood vessels in the abdominal organs.

Here, we describe a visualization and quantification method for murine hind-limb vessels using micro-X-ray computed tomography.

基于计算机断层扫描的小鼠后肢血管三维可视化的样品制备

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ ...

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco&#39;s Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (&#177;4.5) days, with an average cellular yield of 12.3 (&#177; 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (&#177;24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

The present protocol describes an improved methodology for ADSC isolation resulting in a tremendous cellular yield with time gain compared to the literature. This study also provides a straightforward method for obtaining a relatively large number of viable cells after long-term cryopreservation.

从脂肪组织和基质血管分数表征中获取间充质干细胞的技术在长期冷冻保存中的应用

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible ...

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation.
Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes.
The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse- or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

Here, we present a protocol to isolate murine intestinal mesenchyme, including telocytes. These can be used for several applications, such as co-culture with mouse or human-derived organoids, to support growth and better reflect the situation in the original tissue.

小鼠肠间充质分离导致端细胞高产量

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, ...

Preparing Zanba-Stir-Fried Tiebangchui (TBC) Sample for Processing Technology Optimization

To begin a single factor experiment to compare Tiebangchui, or TBC slice thickness, for processing technology optimization, prepare five test groups each having 30 grams of TBC, but different slice thickness. Then weigh 90 grams of Zanba, which is three times as much as that of the TBC in each group. Set the temperature and the time of the automatic stir fry machine to 140 degrees Celsius and 40 minutes respectively.After the machine has heated up to the set temperature, add the weighed amounts of TBC and Zanba into the machine. Then using a high speed smashing machine, grind the Zanba stir-fried TBC into powder. Next, weigh two grams of Zanba stir-fried TBC powder in a conical flask.To the sample in the flask, add three milliliters of ammonia solution followed by 50 milliliters of a mixture of isopropyl alcohol and ethyl acetate, having a volume ratio of one is to one. After recording the weight of the conical flask with the contents, ultrasonicate the TBC sample for 30 minutes at 220 volts and 40 kilohertz maintaining a temperature below 25 degrees Celsius. Weigh the conical flask with the sample once again after the ultrasonic extraction.Make up for the lost weight by adding the isopropyl alcohol and ethyl acetate mixture mentioned previously, and filter the sample solution. Evaporate 25 milliliters of the filtrate to dryness using a rotary evaporator at 40 degrees Celsius. Then dissolve the residue in five milliliters of a solution of 0.05 hydrochloric acid in methanol.Filter the solution through a 0.2 micron syringe filter, and analyze it by performing high performance liquid chromatography, or HPLC. Calculate the contents of the MDA and DDAs in different processing products according to the standard curve. Calculate the comprehensive score based on the results via the critic method.Following the method demonstrated above, compare the amounts of Zanba, processing temperatures, and times for processing technology optimization. The results of the single factor tests revealed that the comprehensive scores of Zanba stir-fried TBC were highest when the sliced thickness is two centimeters, the amount of Zanba is three times, the processing temperature is 120 degrees Celsius, and the processing time is 60 minutes. The results revealed the range of slicing thickness, amount of Zanba, processing temperature, and processing time to be used for future experiments.

制备三叶炒铁棒撀（TBC）样品进行加工技术优化

使用 LC-MS 进行泪液蛋白质组学的非侵入性生物标志物发现

A Protein Suspension-Trapping Sample Preparation for Tear Proteomics by Liquid Chromatography-Tandem Mass Spectrometry

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for several ocular diseases and conditions. The suspension trapping column has been reported to be an efficient and user-friendly sample preparation workflow for the broad application of downstream proteomic analysis. Yet, this strategy has not been well-studied in the analysis of human tear proteome. The present protocol describes an integrated workflow from clinical human tear samples to purified peptides for non-invasive tear protein biomarker research using mass spectrometry, which provides insights into disease biomarkers and monitoring when combined with bioinformatics analysis. A protein suspension trapping sample preparation was applied and demonstrated the discovery of tear proteome with fast, reproducible, and user-friendly procedures, as a universal, optimized sample preparation for human tear fluid analysis. In particular, the suspension trapping procedure outperformed in-solution sample preparation in terms of peptide recovery, protein identification, and shorter sample preparation time.

作者聚焦：使用蛋白质组学研究的标准化方案推进泪液分析 

This protocol describes a method for collecting tear samples using Schirmer strips and an integrated quantitative workflow for discovering non-invasive tear protein biomarkers. The suspension trapping sample preparation workflow enables fast and robust tear sample preparation and mass spectrometric analysis, resulting in higher peptide recovery yields and protein identification than standard in-solution procedures.

液相色谱-串联质谱法制备泪液蛋白质组学的蛋白质悬浮捕获样品

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for ...