通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

摘要

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。

摘要

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.

引言

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 macrophages^5,6. 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.

研究方案

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

1. 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。
2. 制备染色缓冲液（2％胎牛血清（FBS）的在1×Dulbecco氏PBS）中。
3. 加入0.5毫克胶原酶每个毫升盐水的制备胶原酶溶液。
4. 制备氯胺酮/赛拉嗪的麻醉溶液（2:1体积/体积）。

2.肾脏灌注和提取

1. 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。
2. 一旦大鼠被完全麻醉，将其放置在手术台上以仰卧姿势。
3. 应用70％的乙醇于腹部。
4. 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。
5. 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。
6. 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。
7. 放置肾成Hanks'平衡盐溶液中。

3.肾消化细胞悬液

1. 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。
2. 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。
3. 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。
4. 离心在400×g离心15分钟。
5. 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。
6. 离心在400×g离心10分钟。
7. 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。
8. 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。

4.细胞染色和流式细胞仪分析

1. 离心细胞，在100×g离心5分钟。
2. 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。
3. 通过加入1ml染色缓冲液和离心机100 xg的5分钟洗涤。
4. 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）。
5. 在黑暗中再悬浮在抗体混合物20分钟将细胞沉淀，在4℃。
6. 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。
7. 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。
8. 在170 xg离心用1ml透/洗涤缓冲液，离心洗涤2次，每次5分钟。
9. 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。
10. 重悬在CD68抗体溶液中的沉淀，并在黑暗中在4℃孵育50分钟。
11. 用1ml透/洗涤缓冲液，离心洗涤，在170×g离心5分钟。
12. 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。
13. 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。
14. 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。
15. 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。

结果

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...

讨论

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?...

材料

Name	Company	Catalog Number	Comments
Laminar flow hood	Faster		Or equivalent equipment
Centrifuge	Hettich		Or equivalent equipment
Flow cytometer (FACSAria)	BD Biosciences
Fetal bovine serum	BioWest	S1820-500
PBS 10x	LONZA	BE17-515Q
Collagenase	Sigma-Aldrich	12/1/9001
ACK Lysing Buffer	Thermo Fisher Scientific	A10492-01
Flow cytometry strainers	BD Biosciences	340626
Falcon cell strainers	Thermo Fisher Scientific	352340
Flow cytometry tubes	Falcon	352052	5 ml Polystyrene Round-Bottom Tube
Centrifuge tubes	Corning centristar	430791
Water bath	Memmert GmbH + Co. KG	WNE 7	37 °C
Fixation/Permeabilization Solution or Permeabilization/Wash Buffer	BD Biosciences	554714
Rompum (Xylazine)	Bayer		Or equivalent
Ketalar (Ketamine)	Pfizer		Or equivalent
Hanks’ balanced salt solution	Sigma-Aldrich	H8264-500ML
Saline solution	Braun	622415
Anti-CD45 (clone:OX-1) APC-Cy7	Biolegend	202216	Diluted 1:100
Anti-CD68 (clone: ED1) FITC	Bio-RAD	MCA341F	Diluted 35:1,000
anti-CD86 (clone: 24F) PE	Biolegend	200307	Diluted 35:1,000
anti-CD163 (clone: ED2) Alexa Fluor 647	Bio-RAD	MCA342R	Diluted 4:100
Live/dead stain	Molecular Probes	L34955	Diluted 3:1,000