This work was supported by grants from FIS/FEDER (Programa Miguel Servet: CP10/00479, PI13/00802 and PI14/00883), Spanish Society of Atherosclerosis, Spanish Society of Nephrology and Fundaci&#242;n Renal I&#241;igo Alvarez de Toledo (FRIAT) to Juan Antonio Moreno. FIS/FEDER funds PI14/00386 and Instituto Reina Sof&#236;a de Investigaci&#242;n Nefrol&#242;gica to Jes&#250;s Egido. Fundaci&#242;n Conchita Rabago to Melania Guerrero Hue. Fundaci&#242;n Renal I&#241;igo Alvarez de Toledo (FRIAT) to Alfonso Rubio Navarro.

该指令欧洲议会2010/63 / EU和国家指南二千零十三分之五十三下面这个协议被批准由当地机构动物护理和使用委员会。 1.试剂和溶液的制备<ol><li>制备所有的试剂和溶液在无菌条件下并在层流罩下使用。保持在4℃下的解决方案。 </li><li>制备染色缓冲液（2％胎牛血清（FBS）的在1×Dulbecco氏PBS）中。 </li><li>加入0.5毫克胶原酶每个毫升盐水的制备胶原酶溶液。 </li><li>制备氯胺酮/赛拉嗪的麻醉溶液（2:1体积/体积）。 </li></ol> 2.肾脏灌注和提取<ol><li>氯胺酮/甲苯噻嗪腹腔注射（75毫克/ 12毫克/千克体重）麻醉大鼠。仔细捏皮肤的小折，以检查该动物被充分麻醉。然后，盖上眼睛兽医药膏，以防止干燥时的麻醉下。注:4-纹个大的Wistar大鼠在此测定法中使用。 </li><li>一旦大鼠被完全麻醉，将其放置在手术台上以仰卧姿势。 </li><li>应用70％的乙醇于腹部。 </li><li>做一个切口，中央通过腹部皮肤和腹膜，从耻骨到胸腔，暴露胸腔和腹腔。 </li><li>注入盐溶液（0.9％）为腹主动脉灌注直到所有血液从肾脏除去使用灌注系统肾脏。切开主动脉在腹部的水平，以帮助释放血液。 </li><li>从肾门（肾静脉，动脉和输尿管）切割取下大鼠双侧肾脏。为了解封肾脏，用手指，小心地分离胶囊11按下肾脏的边缘。 </li><li>放置肾成Hanks&#39;平衡盐溶液中。 </li></ol> 3.肾消化细胞悬液<ol><li>切半肾用剪刀切成小块，并把片放入1.5 ml管。 注:以下所有浓度为1个样品（1/2大鼠的肾脏）计算。 </li><li>加入1 ml的胶原酶溶液中，并在37℃孵育30分钟。通过颠倒混合，每5分钟，以确保该胶原酶溶液可将整个组织。 </li><li>收集溶液，并使之通过一个过滤器（40微米）配有一个柱塞的帮助，并且在10毫升染色缓冲液重悬它。 </li><li>离心在400×g离心15分钟。 </li><li>重悬在1ml的ACK（氯化铵 - 钾）溶解缓冲液沉淀。 1分钟在室温下30秒后，加入10 mL染色缓冲液以停止反应。 </li><li>离心在400×g离心10分钟。 </li><li>重悬在染色缓冲液（1ml）中的适当体积的沉淀并用过滤器（30微米）过滤细胞悬浮液。 </li><li>计数上使用血球台盼蓝排除细胞的数目和2百万个细胞添加到1.5ml管站都进不去。 </li></ol> 4.细胞染色和流式细胞仪分析<ol><li>离心细胞，在100×g离心5分钟。 </li><li>重悬在100μl大鼠血清的粒料（稀释1:100），10分钟，在4℃下阻断Fc受体。 </li><li>通过加入1ml染色缓冲液和离心机100 xg的5分钟洗涤。 </li><li>混合为细胞表面染色的抗体在100μl染色缓冲液为每个条件:CD45 APC-Cy7的（1:100），CD163 A647（4:100）和溶液来检测活细胞（3:1000）。 </li><li>在黑暗中再悬浮在抗体混合物20分钟将细胞沉淀，在4℃。 </li><li>通过加入1ml染色缓冲液和离心机在100×g离心5分钟洗涤。重复此步骤。 </li><li>在4°C黑暗环境中添加600微升固定/通透的解决方案20分钟。 </li><li>在170 xg离心用1ml透/洗涤缓冲液，离心洗涤2次，每次5分钟。 </li><li>添加CD68 FITC抗体（35:1000）为intracel细胞性染色至100μl透/洗涤缓冲液对各条件。 </li><li>重悬在CD68抗体溶液中的沉淀，并在黑暗中在4℃孵育50分钟。 </li><li>用1ml透/洗涤缓冲液，离心洗涤，在170×g离心5分钟。 </li><li>重悬在100μl透/洗涤缓冲液的沉淀，添加CD86PE抗体（35:1000），并在黑暗中在4℃孵育20分钟。 </li><li>通过加入1ml透/洗涤的，在100×g离心5分钟，缓冲液和离心清洗。 </li><li>重悬在100μl透/洗涤缓冲液的颗粒和把它传递给流式细胞管的流动。 </li><li>通过流式细胞仪分析样品。决定在侧向散射光/前向散射光（SSC / FSC）窗门控活细胞CD45染色。在第二步骤中，分析在CD68 +细胞的CD86和CD163表达。 </li></ol>

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巨噬细胞是发挥不同炎性疾病，包括肾疾病中起重要作用的异构细胞。有在肾脏疾病中的巨噬细胞子集的表征越来越大的兴趣，因为每个细胞巨噬细胞亚群以不同的方式对肾损伤的发展有助于，据报道在肾小球肾炎，糖尿病性肾病和肾癌14-16。在急性肾损伤的早期阶段中，M1巨噬细胞的一个优势是观察，促进肾小管坏死和炎症。然而，在稍后阶段的M2巨噬细胞有较高含量被观察到的解决炎症并参与组织重塑。在慢性肾病，M1和M2的巨噬细胞同时共存在，尽管M1可以是主要的，从而增加了与永久炎性肾损害。因此，重要的是要提高我们的巨噬细胞亚型在肾脏病的病理生理作用的知识。免疫组化研究不能确定在一个可靠的方法17的巨噬细胞亚群的百分比。因此，有必要开发新的技术，以定量地确定不同的巨噬细胞的表型，并了解这些细胞对肾的炎症反应的作用。 该原稿描述的协议通过流式细胞术来确定在大鼠肾脏的巨噬细胞子集的数量和表型。在我们的研究中，大鼠进行醛固酮施用肾损害的炎性模型。在此模型中，我们观察到增加的 CD45 +和C68 +巨噬细胞浸润如通过流式细胞术确定，并通过免疫组织化学证实。此外，M1巨噬细胞（CD68 + / CD86 +），但不M2巨噬细胞的富集，分别在醛固酮处理的小鼠中观察到。这些结果由确定M 1（iNOS和IFN-γ）的mRNA表达和M2巨噬进一步证实年龄标记（的Arg1或IL-10）。隔离协议期间流式细胞仪分析报低细胞死亡率。有几个因素会影响细胞生存力和表面标记检测，例如组织离解的程度以及持续时间和组织消化（酶促，机械等 ）的类型。过度消化增加细胞死亡和巨噬细胞活化，并降低表面标记的表达，而相反的效果可能会从低肾脏离解的结果。然而，当我们的协议在从大鼠腹膜分离巨噬细胞进行的，在这些细胞中观察到CD86和CD163的增强的存在。 我们的协议已被优化，以确定从大鼠醛固酮介导的高血压在肾脏细胞巨噬细胞的异质性，并且因此，可以被外推到其它炎性肾疾病模型。但是，此过程可适于每种实验条件，因为炎症修改组织的完整性，从而影响组织的消化胶原酶率。 要注意的是用于测定总的巨噬细胞含量是很重要的，将细胞固定和透化以允许与CD68抗体18内标记。对于固定的协议包括使用甲醛的可能影响荧光染料缀合的初级抗体，尤其是藻红蛋白。要解决这个问题，除了固定/通透的解决方案，并进一步CD68染色后进行孵育CD86-PE抗体。另一方面，需要注意的是甲醛也可以与可能后的巨噬细胞分离执行的其他测定法，如mRNA表达测定干扰是很重要的。不过，也有可从甲醛固定细胞分离的mRNA商业试剂盒。 总之，该协议描述了肾的巨噬细胞的表型特征的方法在一个quantitati已经使用流式细胞仪的方式。总体而言，这里描述的方案是易于使用，可重复的，和有用的从大鼠肾脏区分不同巨噬细胞群体。

Renal disease is a global health problem, with increased prevalence, and associated with elevated morbidity and mortality1. One of the most important mechanisms involved in the progression and development of renal injury is inflammation, mainly triggered by macrophages. Macrophages play a pivotal role in many inflammatory diseases, including renal disorders2. Thus, an elevated presence of infiltrating macrophages has been reported in biopsies from patients with acute kidney injury (AKI) or chronic kidney disease (CKD)3,4. Recent studies suggest that the long-term outcome of renal disease could be controlled by macrophages5,6. In response to the local microenvironment, macrophages may differentiate into different phenotypes that play diverse biological functions7. Two well differentiated macrophage phenotypes have been established: classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2)8. M1 macrophages promote inflammation, whereas M2 macrophages have an anti-inflammatory role and are involved in tissue repair9. Therefore, a better knowledge of macrophage heterogeneity is necessary to understand their regulation and contribution to renal pathology and develop novel therapeutic approaches. 
Both, murine and rats models have been widely used to understand the molecular and cellular mechanism involved in renal injury10. However, there are substantial differences in the diverse markers used to identify macrophages phenotypes between these rodents. Hence, several murine markers, such as F4/80 or Ly6C are not used in rats, thus limiting the extrapolation of findings between these species. Moreover, there is a limited number of markers describing macrophage phenotypes in rats, explaining the scarce studies analyzing macrophage heterogeneity in these animals as compared with mice. Therefore, new strategies for macrophage subset characterization are necessary to understand the role of macrophages in renal disease models in rats. 
This manuscript describes a protocol for the phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. This technique can be further followed by several assays, including cell sorting and mRNA or protein expression studies to allow in-depth characterization of the role of macrophages in renal disease.

<table border="0" cellpadding="0" cellspacing="0" width="519">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Laminar flow hood</td>
			<td style="width:124px;">Faster</td>
			<td style="width:135px;"></td>
			<td style="width:143px;">Or equivalent equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Centrifuge</td>
			<td style="width:124px;">Hettich</td>
			<td style="width:135px;"></td>
			<td style="width:143px;">Or equivalent equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Flow cytometer (FACSAria)</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:135px;"></td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Fetal bovine serum</td>
			<td style="width:124px;">BioWest</td>
			<td style="width:135px;">S1820-500</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:119px;">PBS 10x</td>
			<td style="width:124px;">LONZA</td>
			<td style="width:135px;">BE17-515Q</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:119px;">Collagenase</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:135px;">12/1/9001</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">ACK Lysing Buffer</td>
			<td style="width:124px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">A10492-01</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Flow cytometry strainers</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:135px;">340626</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Falcon cell strainers</td>
			<td style="width:124px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">352340</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Flow cytometry tubes</td>
			<td style="width:124px;">Falcon</td>
			<td style="width:135px;">352052</td>
			<td style="width:143px;">5 ml Polystyrene Round-Bottom Tube</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Centrifuge tubes</td>
			<td style="width:124px;">Corning centristar</td>
			<td style="width:135px;">430791</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Water bath</td>
			<td style="width:124px;">Memmert GmbH + Co. KG</td>
			<td style="width:135px;">WNE 7</td>
			<td style="width:143px;">37&#186;C</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:119px;">Fixation/Permeabilization Solution or Permeabilization/Wash Buffer</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:135px;">554714</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Rompum (Xylazine)</td>
			<td style="width:124px;">Bayer</td>
			<td style="width:135px;"></td>
			<td style="width:143px;">Or equivalent</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Ketalar (Ketamine)</td>
			<td style="width:124px;">Pfizer</td>
			<td style="width:135px;"></td>
			<td style="width:143px;">Or equivalent</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Hanks&#8217; balanced salt solution</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:135px;">H8264-500ML</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:119px;">Saline solution</td>
			<td style="width:124px;">Braun</td>
			<td style="width:135px;">622415</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:119px;">Anti-CD45 (clone:OX-1) APC-Cy7</td>
			<td style="width:124px;">Biolegend</td>
			<td style="width:135px;">202216</td>
			<td style="width:143px;">Diluted 1:100</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Anti-CD68 (clone: ED1) FITC</td>
			<td style="width:124px;">Bio-RAD</td>
			<td style="width:135px;">MCA341F</td>
			<td style="width:143px;">Diluted 35:1000</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">anti-CD86 (clone: 24F) PE</td>
			<td style="width:124px;">Biolegend</td>
			<td style="width:135px;">200307</td>
			<td style="width:143px;">Diluted 35:1000</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:119px;">anti-CD163 (clone: ED2) Alexa Fluor 647</td>
			<td style="width:124px;">Bio-RAD</td>
			<td style="width:135px;">MCA342R</td>
			<td style="width:143px;">Diluted 4:100</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:119px;">Live/dead stain&#160;</td>
			<td style="width:124px;">Molecular Probes</td>
			<td style="width:135px;">L34955</td>
			<td style="width:143px;">Diluted 3:1000</td>
		</tr>
	</tbody>
</table>

phenotypic characterization of macrophages from rat kidney by flow cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

我们分析了巨噬细胞的异质性与肾脏巨噬细胞浸润的存在增加相关肾损伤的炎症实验模型。在此模型中，肾损伤在饮水中Wistar大鼠3周诱导醛固酮（1毫克-1千克-1天）加的盐（NaCl 1％）的施用，如先前报道12。 我们的实验中的时间表中示出在图1中，首先，肾细胞中分离，采用机械方法和用胶原酶（ 图1A）进一步酶消化。此后，红细胞裂解并通过使用流式细胞仪检测肾巨噬细胞。细胞用抗CD45，CD86和CD163为膜检测和CD68细胞内染色（ 图1B）一起温育。最后，巨噬细胞p的表型根据方案opulations分析显示出图1C。 肾细胞巨噬细胞含量通过与CD45和CD68（ 图2A）双阳性选择细胞进行分析。使用上述协议，增加在醛固酮处理的大鼠中观察到的 CD45 + / CD68 +巨噬细胞相比，对照组（0.57±0.16对0.25±0.02，％CD45 + / CD68每总肾细胞+巨噬细胞）（ 图2，表见插图 ）。 的 CD45 + / CD68 +巨噬细胞的细胞活力，如通过流式细胞术用紫染料染色测定，是常规大于95％。此后，CD86（M1标记物）和CD163（M2标记物）的表达中所评估的（数据未示出） CD45 + / CD68 +细胞。醛固酮增多行政CD45 + / CD68 + / CD86 + M1的含量巨噬细胞（2.87±2.3对1.36±0.68;％ 的 CD45 + / CD68 + / CD86 +每总CD45 + / CD68 +细胞的巨噬细胞）（ 图2）。然而，在这两个醛固酮治疗组和对照组中观察到的 CD45 + / CD68的低编号+ / CD163 + M2巨噬细胞。来分析低CD163表达是否被实验方案中涉及到的蛋白水解脱落，我们重复此过程，包括胶原酶消化，在来自大鼠腹膜分离的巨噬细胞，如前所述13。 如图2B报道，CD45 + / CD68 +巨噬细胞为阳性既CD86和CD163，验证所选择的抗体的效用，并确认我们的协议为M1 / M2巨噬细胞的大鼠肾脏表征的有效性。 为了验证这些结果，与上述实验模型的肾损伤相关的炎症反应通过免疫组织化学和实时PCR分析。如在图3报道，增加在醛固酮+盐处理的大鼠中观察到CD68 +巨噬细胞，与对照组相比。然后，进行了测定巨噬细胞的表型标记物来表征肾M1 / M2巨噬细胞分布。醛固酮+盐处理后观察到的iNOS增加mRNA的表达和IFN-γ（M1标记），而的Arg1或IL-10的mRNA表达量（M2巨噬细胞标记）醛固酮给药后没有改变。总之，这些结果证实，醛固酮+盐给药介导的体内的炎性M1巨噬细胞的表型。 <img alt="图1" src="/files/ftp_upload/54599/54599fig1.jpg" /> 图1:协议肾细胞的分离和巨噬细胞流式细胞术检测。 </stron肾细胞隔离（A）和巨噬细胞的检测用流式细胞仪（B）的g&gt;的示意图。根据不同的分布CD45，CD68，CD86和CD163标志（C）不同的巨噬细胞表型的代表方案。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54599/54599fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54599/54599fig2.jpg" /> 图2:M1和M2巨噬细胞标志物在肾脏从醛固酮通过流式细胞仪时大鼠 CD45染色的代表点图在侧向散射光/前向散射光（SSC / FSC）窗口（ 门控活细胞， 离开了 。 ）从在控制和醛固酮+盐处理的动物的肾脏悬浮液。在散点图内的框标识活细胞表达CD45。包括在这个盒子细胞进行分析以确定CD68 +细胞CD86和CD163表达（A，右 ）。散点图腹膜的巨噬细胞作为用于检测CD45，CD68，CD86和CD163的阳性对照，采用先前所用的相同的策略。该数据以平均值±SEM（B）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54599/54599fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54599/54599fig3.jpg" /> 图3:醛固酮+盐务管理局后炎症反应。 （A）CD68染色和的代表性图像（B）中通过RT-PCR对M1标记（iNOS和INFγ）和M2标记（的Arg1和IL-10）的控制和醛固酮+盐处理的大鼠测量mRNA水平的定量分析。数据速递ED平均±SEM。 * P &lt;0.05与对照。比例尺:100微米<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54599/54599fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry at JoVE.com

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

该原稿描述的表型，并通过流式细胞从大鼠肾脏定居巨噬细胞的定量分析的详细协议。所得染色的细胞也可以用于其它应用，包括细胞分选，基因表达分析或功能性研究，从而增加在实验模型中获得的信息。

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

phenotypic-characterization-macrophages-from-rat-kidney-flow

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

14.6K 次观看.  Autonoma University. 该原稿描述的表型，并通过流式细胞从大鼠肾脏定居巨噬细胞的定量分析的详细协议。所得染色的细胞也可以用于其它应用，包括细胞分选，基因表达分析或功能性研究，从而增加在实验模型中获得的信息。 

视频: Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

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 ...

科研

免疫与感染

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

考试流式细胞仪检测胸腺阳性和阴性选择

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征:两个单核细胞分离方法的比较

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

人肺树突状细胞:空间分布与表型鉴定支气管活检采用免疫组化和流式细胞仪

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

通过流式细胞术分析来自中枢神经系统的小胶质细胞和单核细胞衍生的巨噬细胞

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

双氟铬流式细胞仪对七种免疫细胞子集的判别

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...