这项工作得到了资金支持fromthe比利时科学研究基金（FNRS），莱斯阿美族DE L&#39;研究所博尔德，FNRS操作Télévie，比利时计划癌症，全宗朗博-Marteaux，全宗JC Heuson和全宗Barsy。

注：使用经研究所朱尔·博尔代的医学伦理委员会与每个病人获得书面知情同意的协议被收购所有标本。 1.制备组织匀浆<ol><li>解剖手术切除组织（恶性和正常组织从手术室切除）是在病理实验室由受过训练的人员立即拾取。肿瘤，NANT（取最远的距离肿瘤的可能），并在1个正常组织碎片常规处理 - 3小时手术切除使用标准的生物安全程序，人体组织在BSL2实验室。该协议的流程图被示于图1。 </li><li>权衡所有组织碎片（正常，NANT和肿瘤），测量的长度，宽度，和高度（长度×宽度×高度）。这对于细胞亚群的随后正常化的一个重要步骤，提取的RNA， 等等。 NOTE：样本量的范围为100至10,000 mm 3的无脂 ​​肪如果可能的话。 </li><li>印上与H＆E染色载玻片的肿瘤片段，以验证该组织实际上是肿瘤的一部分。 <ol><li>通过按肿瘤片段显微镜载玻片和应用温和的压力，用手指几秒钟做到这一点。 </li><li>修复用异丙醇进行2分钟，随后在水中的洗涤步骤的幻灯片。染液组织30秒，Mayer的苏木。 </li><li>在水6浴洗幻灯片。在桃红B染色的2％，持续15秒。 </li><li>在水一浴跟四个浴池异丙醇和完成与水一浴洗。 </li><li>孵育在异丙醇1分钟和漏。清除二甲苯的两个浴室。 </li><li>安装用二甲苯基于安装中。检查对肿瘤细胞结构（ 图2）。 注：主要，肿瘤细胞坚持印记幻灯片 - 间质，淋巴或广告ipose细胞很少残留，而使肿瘤细胞之间的空间（ 图2）。 </li></ol></li><li>放置在含有1ml的化学上确定的，无血清造血细胞培养基的小培养皿所述组织片段（以下简称为培养基）在室温和切块成小块（约1 - 2平方毫米 ），使用无菌的手术刀。 </li><li>转让的一切（组织碎片+介质）的机械离解Ç管。 </li><li>冲洗培养皿并手术刀用2ml使用巴氏吸管介质的，这增加了C管（培养基体积为离解= 3毫升）中。 </li><li>使用机械离解程序A.01对于C管（最温和的程序），以均匀的组织碎片成单细胞悬浮液。放置了C管中的装置和连续运行该程序两次（一个周期= 25秒）。 注意：此均质过程已经建立和验证人类乳房组织，其它匝或者或组织类型可能需要使用不同的程序，应首先进行测试。 </li><li>从装置删除C管，并直接倒出匀浆成40μm的细胞过滤器安置在一个50ml管中。使用相同的巴斯德吸管如在步骤1.6，转移剩余在C管的细胞过滤的任何液体。 </li><li>过滤后的液体转移到一个15毫升的管中，使用1毫升微量移液器尖端。暂时保持细胞过滤和50ml管中。 </li><li>冲洗了C管与另外3毫升培养基并传送此，再次使用相同的巴斯德吸管如在步骤1.6，该细胞过滤仍然落座于50ml管中。通过轻轻地移动它周围的过滤器，用干净巴斯德吸管或1毫升提示，随后丢弃，以避免污染洗脱液挤被困在unhom​​ogenized组织进50毫升管中的残留液体的最大量。 </li><li>将细胞过滤对T倒挂他原来的C管和漂洗用3ml培养基，使得unhom​​ogenized组织降回到C管。 </li><li>再均匀化作为用于A.01程序的两个周期的步骤1.7。 </li><li>倾通过细胞过滤网落座于50ml管中该第二匀浆，再次冲洗了C管用3ml培养基（如在步骤1.10），并使用巴斯德吸管转移到细胞过滤落座于50ml管中再次挤压的最大从残留的结缔组织截留在细胞过滤的液体量。 </li><li>在这一点上，约2.5毫升的体积是在15ml试管和〜9毫升50毫升管。 </li></ol> 2.分离的组织上清和细胞<ol><li>离心在15毫升匀浆和50ml试管15分钟，在600×g离心，在室温下进行。 </li><li>滗从15毫升管中的SN到干净的管中并暂时储存在4℃。这上清=原发肿瘤，NANT或正常TIssue SN（最终体积2.5ml）中，随后澄清并在-80℃下以供将来分析（见下文）在存储之前等分。 </li><li>丢弃从50ml管中的上清液。 </li><li>在1毫升培养基中的终体积轻轻悬浮两种细胞粒料。简要地说，首先轻轻（轻敲管上的硬表面）打破在两个管中的细胞沉淀。重悬松散细胞沉淀在50ml的用500μl培养基中，该细胞悬浮液转移到15毫升管重新悬浮所述第二颗粒。与第二500μl的培养基中对细胞的最大回收重复此步骤一次。 </li><li>转移10微升细胞悬浮液至小管中，混合10微升台盼蓝（稀释1：1），并计算使用血球存活细胞的数目。 注意：在这一点上的细胞悬浮液的一小部分，也可以通过流式细胞术分析来评估细胞大小，粒度并且如果需要的亚群的标记有限数量的更多PREC在匀浆中相对细胞分布之前广泛分析或实验伊势评估。所有的分析流式细胞仪结合CD45标签的亚正常化。 </li><li>通过离心沉淀细胞，在300×g离心10分钟，在室温下。从肿瘤，NANT，或正常组织的细胞是现在已准备好用于进一步纯化或分析。这些额外的步骤是最好在同一天进行的外科手术时。 注：对于流式细胞仪分析，但不是细胞分选的残余红细胞应抗体标记后，加入0.4红血细胞裂解缓冲液，以细胞沉淀，立即为1秒涡旋并温育至少10分钟，在室温进行裂解温度（避光）分析之前。 </li></ol> 3.澄清组织的上清液<ol><li>离心1.5 mL管与组织SN在15000 XG为15分钟，在4℃。 </li><li>小心地取出吨他上清液不接触或干扰球。转移到一个干净的试管（或管），这取决于所述数量和期望的等分试样的体积。 </li><li>存储上清液在-80℃下以供将来使用。 </li></ol> 4.患者血<ol><li>收集从每个病人作为对照通过静脉穿刺到肝素化管中的前一天，以外科手术的血液样品。离心血液于400×g离心10分钟，在20ºC与制动切断以获得血浆和血沉棕黄层。 </li><li>取出血浆，并通过离心，在20ºC澄清它以10,000×g离心15分钟。等分试样并储存血浆在-80℃下以供将来使用。淡化捉鬼外套中等</li><li>分离使用标准聚蔗糖 - 泛影葡胺密度梯度离心之前立即进行分析，亚群分离，DNA / RNA /蛋白质的提取或超低温保存的单核细胞。 </li></ol> 5.流式细胞仪<ol><li>根据制造标签的细胞的r指令在4℃，避光。裂解红血细胞在从抗体标记后的组织碎片的细胞悬浮液通过加入0.4毫升的细胞裂解缓冲液，以细胞沉淀。 </li><li>涡旋立即1秒和孵化至少在室温下10分钟，避光。通过在流式细胞仪进行数据采集，而不洗涤标记的细胞。 </li></ol>

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The authors declare that no conflict of interest exists.

该研究描述了在正常和恶性乳腺组织匀浆中迅速地制备无酶消化用于随后的细胞分选，提取，冷冻保存和/或CD45 +亚群的表型分析的优化方法。本实验方法的目标是产生紧密反映其在体内的状态，将它们与从手术室新鲜组织的最低限度的操作比较正常组织的TIL的图像。到今天为止，我们的实验室已使用此协议分析&gt; 250新鲜BC组织（肿瘤和NANT），&gt; 35正常乳腺组织，从乳腺减少。原?...

The tumor microenvironment is composed of various cell types with numerous studies showing they each play distinct and important roles in tumorigenesis1,2. These include, but are not limited to, infiltrating immune cells, stromal cells, endothelial cells and tumor cells3. Ex vivo studies of tumor infiltrating lymphocytes (TIL; CD45+ cells or leukocytes, which are predominantly lymphocytes in breast tumors) from fresh human tissue samples is made difficult by their low frequency, the small sample sizes often available for research and the potential for loss of viability during extraction. Because immune cells infiltrating tumors are usually present as passengers rather than permanent residents in general they are easier to release from the tissue matrix.
Dissociating tumor tissue while maintaining cellular integrity is technically challenging and has traditionally been performed using a combination of mechanical and enzymatic steps to prepare single cell suspensions4-8. This approach involves lengthy incubation periods and is associated with a significant reduction in cell viability as well as the loss of cell surface receptors by enzymatic cleavage. High quality flow cytometric studies characterizing TIL in the tumor microenvironment as well as clean purifications of CD45+ subpopulations by flow cytometry or antibody-coated beads are more difficult to achieve from enzyme-digested tumor tissue. In addition, the supernatant (SN) from the resulting tumor homogenate is not amenable to further analysis including quantification of secreted proteins (cytokines, chemokines, immunoglobulins or tumor antigens) or experimental treatment of normal cells, because of the potential for protein degradation in the enzymatic digests.
In our search for a method to prepare single cell homogenates from breast tissues [including tumor, non-adjacent non-tumor (NANT) and normal (from mammary reductions) breast tissues] without enzymatic digestion, we tested a variety of mechanical homogenization techniques. Homogenates prepared using a mechanical dissociator had increased cell viability (2-fold) and total cell recovery (2-fold) while preserving surface receptor expression. Enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells suggesting they were all released in the initial homogenate. Thus, this rapid and simple approach allows both qualitative and quantitative assessment of the CD45+ subpopulations present in various normal and malignant human tissues. An added advantage of this approach is that the SN from the initial homogenate (primary tissue SN) can be collected and stored for further analysis or experimentation.

<table border="0" cellpadding="0" cellspacing="0" width="980">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Equipment</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:409px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">GentleMacs Dissociator&#160;</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-093-235</td>
			<td>BD Medimachine is somewhat equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Centrifuge 5810 R</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard table top centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Centrifuge 5417 R</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard microcentrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Esco Class II A2 Biosafety Cabinet</td>
			<td>ESCO global</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard BSL2 hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Inverted Microscope</td>
			<td style="width:163px;">Nikon eclipse TS100</td>
			<td style="width:137px;">N/A</td>
			<td>or other microscope compatible for a hemacytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">B&#252;rker Chamber</td>
			<td style="width:163px;">Marienfield&#160;</td>
			<td style="width:137px;">640210</td>
			<td>or other standard hemacytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Navios Flow Cytometer</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">N/A</td>
			<td>or other flow cytometer (8-10 color recommended)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;"></td>
			<td style="width:163px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Materials</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">GentleMacs C-Tube</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-344</td>
			<td>BD Medimachine uses Filcon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Cell Culture Dish</td>
			<td style="width:163px;">Sarstedt</td>
			<td style="width:137px;">72,710</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Disposable Scalpel</td>
			<td style="width:163px;">Swann-Morton</td>
			<td style="width:137px;">0510</td>
			<td>or standard single use sterile scalpel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Cell Strainer 40&#181;m</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">734-0002</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Falcon Tube 50mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352070</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Falcon Tube 15mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352097</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD FACS Tube 5mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352008</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sterile Pasteur Pipette 5 mL&#160;</td>
			<td style="width:163px;">VWR</td>
			<td style="width:137px;">612-1685</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Microfuge Tube 1.5 mL</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">7805-00</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;"></td>
			<td style="width:163px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Reagents</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">X-Vivo 20</td>
			<td style="width:163px;">Lonza</td>
			<td style="width:137px;">BE04-448Q</td>
			<td>serum-free medium recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Phosphate buffered saline</td>
			<td style="width:163px;">Lonza</td>
			<td style="width:137px;">BE17-516F</td>
			<td>standard physiological PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Trypan blue&#160;</td>
			<td style="width:163px;">VWR</td>
			<td style="width:137px;">17942E</td>
			<td>or other vital stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">VersaLyse</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">A09777</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fixable viability Dye eFluor 780&#160;</td>
			<td style="width:163px;">eBioscience</td>
			<td style="width:137px;">65-0865-14</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD3 FITC</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345763</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD3 Vio Blue</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-094-363</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD4 PE</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345769</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD4 APC</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-091-232</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD8 ECD</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">737659</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD8 PerCP</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345774</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD19 APC-Vio770</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-643</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD45 VioGreen</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-906</td>
			<td>for flow cytometry experiments</td>
		</tr>
	</tbody>
</table>

a simple and rapid protocol to non-enzymatically dissociate fresh human tissues for the analysis of infiltrating lymphocytes

恶性细胞逃避免疫系统，从先天性和适应性免疫应答特征为肿瘤逃逸的能力现在被接受为癌症的重要标志。我们对乳癌的研究主要集中在积极的作用，肿瘤浸润淋巴细胞肿瘤进展和患者预后发挥。为了实现这一目标，我们制定了正常和异常组织完整淋巴样细胞的快速分离的方法，努力以评估他们接近他们的原生状态。匀浆用机械离解秀既增加了活力和细胞的恢复，同时保持表面受体的表达相比，酶消化的组织准备。另外，剩余的不溶物的酶消化未恢复额外的CD45 +细胞表示在初级匀浆的定量和定性测量可能真正反映在组织fragm浸润亚耳鼻喉科。在淋巴样细胞中这些匀浆可以容易地使用免疫（表型，细胞增殖等 ）或分子（DNA，RNA和/或蛋白质），其特征接近。 CD45 +细胞也可以用于亚群纯化， 体外扩增或冷冻保存。这种方法的一个额外的好处是，从匀浆主组织上清液可用于表征和比较细胞因子，趋化因子，免疫球蛋白和抗原存在于正常和恶性组织。这个协议功能非常好为人类乳房组织和应适用于各种各样的正常和异常组织。

组织碎片用可商购的组织离解的溶液或胶原酶的各种实验室的混合物，DNA酶和/或透明质酸酶抑制剂酶消化，切割各种各样的受体的细胞的表面上。我们的研究中，最初集中在CD4 + T细胞浸润乳腺肿瘤，很快出现一个主要的技术问题，是由于使用标准酶消化协议4-8表面的CD4受体的裂解。我们测试了多种胶原酶的酶有或无DNA酶和透明质酸酶抑制剂，发现胶原酶I和II完全从细胞表面除去?...

Watch this Scientific Journal Video about A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes at JoVE.com

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

a-simple-rapid-protocol-to-non-enzymatically-dissociate-fresh-human

Research

JoVE Journal

Immunology and Infection

19.9K 次观看.  Université Libre de Bruxelles. This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

视频: A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

一个简单的的Chelex协议对DNA的提取<em&gt;按蚊</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

一个简单的协议介导的血小板聚集<em&gt;恶性疟原虫</em&gt;受感染的红细胞在一个资源匮乏的设置

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

从人类腭扁桃体B和T淋巴细胞有效隔离协议

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

对于血液处理快速的方法，以提高血浆肽水平的人血产量

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

一种优化的协议分析和糖酵解线粒体呼吸的淋巴细胞

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

T淋巴细胞有丝分裂原诱导芽生的快速定量辨识免疫调节药物

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...