Name: phenotypic characterization of macrophages from rat kidney by flow cytometry
Uploaded: 2016-10-18T14:00:00
Description: Watch this Scientific Journal Video about Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry at JoVE.com

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?...

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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?...

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.

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		<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...

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

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