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Isolation and Analysis of Stromal Vascular Cells from Visceral Adipose Tissue
.内脏脂肪组织来源基质血管细胞的分离和分析   

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Abstract

The obesity epidemic is the underlying driver of the type 2 diabetes mellitus epidemic. A remarkable accumulation of various pro-inflammatory immune cells in adipose tissues is a hallmark of obesity and leads to pathogenesis of tissue inflammation and insulin resistance. Here, we describe a detailed protocol to isolate adipose tissue stromal vascular cells (SVCs), which enrich various immune cells of adipose tissues. These SVCs can be used to examine the population and activation status of immune cells by tracking their cell surface antigens, gene expression, and activation of specific signaling pathways.

Keywords: Adipose tissue(脂肪组织), Stromal vascular cell(基质血管细胞), Collagenase digestion(胶原酶消化), Immune cell(免疫细胞), Flow cytometry analysis(流式细胞仪分析)

Background

Over the past several decades, obesity is now an epidemic and has become one of the most common causes of insulin resistance. Insulin resistance is the key etiology for the pathogenesis of metabolic syndrome. Prolonged status of metabolic syndrome drives the development of type 2 diabetes mellitus (T2DM) (Romeo et al., 2012; Johnson and Olefsky, 2013; Saltiel and Olefsky, 2017).

Chronic low-degree tissue inflammation, accompanied by enhanced immune cell infiltration, is a hallmark of obesity in both rodent and human and is a major causal factor for the pathogenesis of insulin resistance through promoting the inflammation status and interrupting the insulin signalling (Romeo et al., 2012; Johnson and Olefsky, 2013; Saltiel and Olefsky, 2017). The infiltrated immune cells such as pro-inflammatory macrophages and B cells play critical roles in modulating obesity-associated adipose tissue inflammation and insulin resistance (Weisberg et al., 2003; Winer et al., 2011). Chronic nutrient excess drives adipose tissue macrophages (ATMs) to undergo a unique phenotypic switch from anti-inflammatory M2-like activation in lean adipose tissue to a more pro-inflammatory M1-like activation state in obese tissues (Lumeng et al., 2007; Nguyen et al., 2007; Lumeng et al., 2008). Pro-inflammatory M1-like ATMs contribute to the development of tissue inflammation and systemic insulin resistance in obesity. Our recent study also demonstrates that leukotriene B4 (LTB4)-induced recruitment and activation of adipose tissue B2 (ATB2) cells can cause obesity-induced insulin resistance (Ying et al., 2017). In this protocol, we provide a step-by-step procedure to isolate stromal vascular cells from adipose tissue and characterize various immune cells in adipose tissues.

Materials and Reagents

  1. Pipette tips (USA Scientific)
  2. 100-mm Petri dish
  3. 50 ml Falcon tube (Corning, Falcon®, catalog number: 352070 )
  4. Nylon biopsy bag (Electron Microscopy Sciences, catalog number: 62324-35 )
  5. MicroAmp Optical 96-well reaction plate (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: N8010560 )
  6. Stromal vascular cells (SVCs)
  7. 70% ethanol
  8. BDTM stabilizing fixative buffer (BD, BD Biosciences, catalog number: 339860 )
  9. Phosphate-buffered saline (PBS)
  10. 2% fetal bovine serum (FBS)
  11. Antibody
    1. Rabbit monoclonal anti-GAPDH (Cell Signaling Technology, catalog number: 5174 )
    2. Rabbit monoclonal anti-Phospho-NF-κB p65 ( Cell Signaling Technology , catalog number: 3033 )
    3. PE-Cyanine7 anti-mouse F4/80 (Thermo Fisher Scientific, eBioscienceTM, catalog number: 25-4801-82 )
    4. Alexa Fluor 488 anti-mouse CD11b (Thermo Fisher Scientific, eBioscience TM, catalog number: 53-0112-82 )
    5. APC anti-mouse CD11c (Thermo Fisher Scientific, eBioscienceTM , catalog number: 17-0114-82 )
    6. PE anti-mouse CD206 ( BioLegend , catalog number: 141706 )
    7. eVolve-605 anti-mouse CD45 (Thermo Fisher Scientific, eBioscienceTM, catalog number: 83-0451-42 )
    8. APC anti-mouse CD19 (Thermo Fisher Scientific, eBioscienceTM , catalog number: 17-0193-82 )
  12. Trizol reagent (Thermo Fisher Scientific, InvitrogenTM , catalog number: 15596026 )
  13. Direct-zol RNA kits (Zymo Research, catalog number: R2070 )
  14. High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4368813 )
  15. qPCR primers (Table 1)

    Table 1. qPCR primer information


  16. SYBR Green PCR Master mix (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4309155 )
  17. Hanks’ balanced salt solution (HEPES) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  18. Collagenase II (Sigma-Aldrich, catalog number: C1764-50MG )
  19. Bovine serum albumin (BSA)
  20. Ammonium chloride (NH4Cl)
  21. Potassium bicarbonate (KHCO3)
  22. 5% EDTA
  23. Sodium azide (NaN3)
  24. Iscove’s Modified Dulbecco’s Medium (IMDM)
  25. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number. 15140122 )
  26. Digestion buffer (see Recipes)
  27. Red blood cell lysis buffer (see Recipes)
  28. FACS staining buffer (see Recipes)
  29. Complete culture medium (see Recipes)

Equipment

  1. Pipettes
  2. Mortar and pestle
  3. Curved scissor
  4. New Brunswick Scientific 12400 incubator shaker (Eppendorf, model: New BrunswickTM 124 )
  5. Eppendorf centrifuge 5810R (Eppendorf, model: 5810 R )
  6. TC20 automated cell counter (Bio-Rad Laboratories, model: TC20TM, catalog number: 1450102 )
  7. BD FACSCanto flow cytometry analyzer
  8. StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Applied BiosystemsTM, model: StepOnePlusTM , catalog number: 4376600)
  9. DNA Engine Peltier Thermal Cycler (Bio-Rad Laboratories, model: PTC-200 )
  10. NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, model: NanoDropTM 1000 )

Software

  1. FlowJo
  2. GraphPad Prism

Procedure

  1. Isolation of stromal vascular cells (SVCs) from adipose tissue (Figure 1)


    Figure 1. Scheme for isolation of stromal vascular cells from adipose tissue

    1. Male mice with 18-20 weeks age were euthanized following the ICCUAC approval protocol.
    2. After thoroughly wet the fur with 70% ethanol and then open the thoracic cavity, epididymal fat tissues were collected and weighed.
    3. Mince fat tissues on a 100-mm Petri dish by using a curved scissor.
    4. Transfer minced tissues to a 50 ml Falcon tube containing digestion buffer (HBSS containing 2% BSA and 100 mM HEPES).
    5. Add 10 ml digestion buffer (see Recipes) per 1 g fat tissue.
    6. Incubate in an incubator shaker (secure the tubes horizontally) at 220 rpm for 10 min, 37 °C (no chunk tissue represents complete digestion).
    7. Add equal volume of complete culture medium and mix to cease the digestion.
    8. Filter through a nylon biopsy bag (pore size = 250 µm) into a new 50 ml Falcon tube.
    9. Centrifuge at 1,500 x g at 4 °C for 10 min.
    10. Decant the supernatant and re-suspend cell pellet in 1 ml of PBS/2% FBS.
    11. Add 2 ml of red blood cell lysis buffer (see Recipes) (1:2) and mix.
    12. Incubate on ice for 10 min.
    13. Add 3 ml of complete medium (1:1) and mix.
    14. Centrifuge at 1,000 x g for 5 min, 4 °C.
    15. Decant the supernatant.
    16. SVCs are resuspended with 0.5 ml of PBS/2% FBS and then counted using the TC20 automated cell counter. Cell concentration is adjusted to 2 x 106 cells per ml, and the cells are ready for the downstream experiments.

  2. Flow cytometry analysis
    1. Prepare 0.5-1 x 106 SVCs and resuspend in FACS staining buffer (see Recipes).
    2. Stain SVCs with the fluorescent-conjugated antibodies (Materials and Reagents #11) following the manufacture’s instruction.
    3. Incubate for 15 min at RT (light sensitive; keep in dark).
    4. Add 1 ml FACS staining buffer.
    5. Centrifuge at 1,000 x g for 5 min, 4 °C.
    6. Decant the supernatant.
    7. Re-suspend with 100-200 µl FACS staining buffer.
      Option: Cells can be stored in BDTM stabilizing fixative buffer up to 3 days at 4 °C and then used for FACS analysis.
    8. Then the sample is ready for flow cytometry analysis (Figure 2) or specific immune cell isolation by cell sorter.


      Figure 2. Flow cytometry analysis of immune cells of visceral adipose tissue (VAT). The SVCs were stained with fluorescent-conjugated antibodies against CD11b, F4/80, CD11c, CD206, CD19, and then examined by flow cytometry analysis. A. Adipose tissue macrophages (ATMs) were defined as CD11b+F4/80+ subpopulations and displayed as percentage of CD45+ cells. B. M1 and M2 ATMs were defined as CD11b+F4/80+CD11c+CD206- and CD11b+F4/80+CD11c-CD206+, respectively. These cell populations were shown as percentage of ATMs. C. B cells were defined as CD45+CD19+ and the cell population was presented as the percentage of CD45+ cells.

  3. RT-PCR
    1. SVCs are resuspended in Trizol (add 100 µl Trizol reagent to < 105 cells; add 300 µl Trizol reagent to < 106 cells) and incubated for 5 min at room temperature (RT).
    2. After centrifugation at 1,000 x g for 5 min (RT), transfer the supernatant to a new tube.
    3. Total RNAs are isolated using Direct-zol RNA kits.
    4. RNA concentration is measured by NanoDrop 1000 spectrophotometer.
    5. RNAs (500 ng) are converted to cDNA using High-Capacity cDNA Reverse Transcription Kit.
    6. The reverse transcription reactions are under the thermal cycling program: 25 °C for 10 min, then 37 °C for 120 min, then 85 °C for 5 min, and then 4 °C.
    7. The cDNA is mixed primers and SYBR Green PCR Master Mix in the MicroAmp Optical 96-well reaction plate.
    8. The real time PCR (qPCR) reaction is ran under the thermal cycling condition: 95 °C for 10 min, then 40 cycles (95 °C for 15 sec, and then 60 °C for 1 min).
    9. The RT-PCR results show that lean SVCs exhibit greater arginase 1 expression but less TNFα abundance than obese SVCs (Figures 3A and 3B).


      Figure 3. Inflammatory status of SVCs by RT-PCR analysis and Western blots. There was less arginase 1 (A) but more TNFα (B) abundance in obese SVCs, compared to the lean SVCs (qPCR primer information see Table 1). C. Activation of NF-κB signalling pathway of SVCs (without stimulation) was evaluated by Western blotting analysis (Antibodies information see Materials and Reagents #11). Data are presented as mean ± SEM. n = 3 per group. **P < 0.01, ***P < 0.001, Student’s t-test.

  4. Western blots
    See previous article by Zhang, 2011. The Western blotting analysis indicates that obese SVCs have higher level of phosphorylated P65 than lean SVCs (Figure 3C).

Recipes

  1. Digestion buffer (prepare freshly), 50 ml
    50 ml HBSS
    50 mg collagenase II
    100 mg BSA
    100 mM HEPES
  2. Red blood cell lysis buffer (10x), 1 L
    PBS (without calcium and magnesium)
    8.3 g ammonium chloride (NH4Cl) (150 mM)
    1.0 g potassium bicarbonate (KHCO3) (10 mM)
    1.8 ml of 5% EDTA (0.1 mM)
  3. FACS staining buffer, 100 ml
    98 ml PBS (without calcium and magnesium)
    2 ml FBS
    0.1 g NaN3
  4. Complete culture medium
    500 ml Iscove’s modified Dulbecco’s medium (IMDM)
    50 ml FBS
    5 ml penicillin-streptomycin (Thermo Fisher Scientific)

Acknowledgments

This protocol was adapted from Ying et al. (2017). This study was funded by the American Heart Association (16POST31350039 to W. Ying), the National Natural Science Foundation of China (81600610 to W. Ying).

References

  1. Johnson, A. M. and Olefsky, J. M. (2013). The origins and drivers of insulin resistance. Cell 152(4): 673-684.
  2. Lumeng, C. N., DelProposto, J. B., Westcott, D. J. and Saltiel, A. R. (2008). Phenotypic switching of adipose tissue macrophages with obesity is generated by spatiotemporal differences in macrophage subtypes. Diabetes 57(12): 3239-3246.
  3. Lumeng, C. N., Deyoung, S. M., Bodzin, J. L. and Saltiel, A. R. (2007). Increased inflammatory properties of adipose tissue macrophages recruited during diet-induced obesity. Diabetes 56(1): 16-23.
  4. Nguyen, M. T., Favelyukis, S., Nguyen, A. K., Reichart, D., Scott, P. A., Jenn, A., Liu-Bryan, R., Glass, C. K., Neels, J. G. and Olefsky, J. M. (2007). A subpopulation of macrophages infiltrates hypertrophic adipose tissue and is activated by free fatty acids via Toll-like receptors 2 and 4 and JNK-dependent pathways. J Biol Chem 282(48): 35279-35292.
  5. Romeo, G. R., Lee, J. and Shoelson, S. E. (2012). Metabolic syndrome, insulin resistance, and roles of inflammation–mechanisms and therapeutic targets. Arterioscler Thromb Vasc Biol 32(8): 1771-1776.
  6. Saltiel, A. R. and Olefsky, J. M. (2017). Inflammatory mechanisms linking obesity and metabolic disease. J Clin Invest 127(1): 1-4.
  7. Weisberg, S. P., McCann, D., Desai, M., Rosenbaum, M., Leibel, R. L. and Ferrante, A. W., Jr. (2003). Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 112(12): 1796-1808.
  8. Winer, D. A., Winer, S., Shen, L., Wadia, P. P., Yantha, J., Paltser, G., Tsui, H., Wu, P., Davidson, M. G., Alonso, M. N., Leong, H. X., Glassford, A., Caimol, M., Kenkel, J. A., Tedder, T. F., McLaughlin, T., Miklos, D. B., Dosch, H. M. and Engleman, E. G. (2011). B cells promote insulin resistance through modulation of T cells and production of pathogenic IgG antibodies. Nat Med 17(5): 610-617.
  9. Ying, W., Wollam, J., Ofrecio, J. M., Bandyopadhyay, G., El Ouarrat, D., Lee, Y. S., Oh, D. Y., Li, P., Osborn, O. and Olefsky, J. M. (2017). Adipose tissue B2 cells promote insulin resistance through leukotriene LTB4/LTB4R1 signaling. J Clin Invest 127(3): 1019-1030.
  10. Zhang, H. (2011). Western blot for detecting phosphorylated STAT3. Bio Protoc Bio101: e125.

简介

肥胖流行是2型糖尿病流行病的根本驱动因素。 脂肪组织中各种促炎免疫细胞的显着积累是肥胖的标志,并导致组织炎症和胰岛素抵抗的发病机制。 在这里,我们描述了分离脂肪组织基质血管细胞(SVCs)的详细方案,其丰富了脂肪组织的各种免疫细胞。 这些SVC可用于通过跟踪其细胞表面抗原,基因表达和特异性信号通路的激活来检查免疫细胞的群体和激活状态。
【背景】过去几十年来,肥胖现在已成为一种流行病,已成为胰岛素抵抗最常见的原因之一。胰岛素抵抗是代谢综合征发病机理的关键病因。代谢综合征的延长状态推动了2型糖尿病(T2DM)的发展(Romeo et al。,2012; Johnson and Olefsky,2013; Saltiel and Olefsky,2017)。
   慢性低度组织炎症伴随着增强的免疫细胞浸润,是啮齿动物和人类肥胖症的标志,并且是通过促进炎症状态和中断胰岛素信号传导来促进胰岛素抵抗的发病机制的主要因素(Romeo等2012年; Johnson和Olefsky,2013; Saltiel和Olefsky,2017)。浸润的免疫细胞如促炎性巨噬细胞和B细胞在调节肥胖相关脂肪组织炎症和胰岛素抵抗中起关键作用(Weisberg等,2003; Winer等,2011)。慢性营养过剩驱使脂肪组织巨噬细胞(ATMs)经历独特的表型转换,从瘦脂肪组织中的抗炎M2样活化转变为肥胖组织中更致炎的M1样活化状态(Lumeng等,2007; Nguyen等人,2007; Lumeng等人,2008)。促炎症M1样ATM有助于肥胖组织炎症和全身胰岛素抵抗的发展。我们最近的研究还表明,白三烯B4(LTB4)诱导脂肪组织B2(ATB2)细胞的募集和激活可引起肥胖诱导的胰岛素抵抗(Ying等,2017)。在该方案中,我们提供了一个逐步的过程,从脂肪组织中分离出基质血管细胞,并表征脂肪组织中的各种免疫细胞。

关键字:脂肪组织, 基质血管细胞, 胶原酶消化, 免疫细胞, 流式细胞仪分析

材料和试剂

  1. 移液器提示(美国科学)
  2. 100毫米培养皿
  3. 50ml Falcon管(Corning,Falcon ®,目录号:352070)
  4. 尼龙活检袋(Electron Microscopy Sciences,目录号:62324-35)
  5. MicroAmp光学96孔反应板(Thermo Fisher Scientific,Applied Biosystems TM,目录号:N8010560)
  6. 基质血管细胞(SVCs)
  7. 70%乙醇
  8. 稳定固定缓冲液(BD,BD Biosciences,目录号:339860)
  9. 磷酸盐缓冲盐水(PBS)
  10. 2%胎牛血清(FBS)
  11. 抗体
    1. 兔单克隆抗GAPDH(Cell Signaling Technology,目录号:5174)
    2. 兔单克隆抗磷酸化NF-κBp65(Cell Signaling Technology,目录号:3033)
    3. PE-Cyanine7抗鼠F4 / 80(Thermo Fisher Scientific,eBioscience TM,目录号:25-4801-82)
    4. Alexa Fluor 488抗小鼠CD11b(Thermo Fisher Scientific,eBioscience TM,目录号:53-0112-82)
    5. APC抗小鼠CD11c(Thermo Fisher Scientific,eBioscience TM,目录号:17-0114-82)
    6. PE抗小鼠CD206(BioLegend,目录号:141706)
    7. eVolve-605抗小鼠CD45(Thermo Fisher Scientific,eBioscience TM,目录号:83-0451-42)
    8. APC抗小鼠CD19(Thermo Fisher Scientific,eBioscience TM,目录号:17-0193-82)
  12. Trizol试剂(Thermo Fisher Scientific,Invitrogen TM,目录号:15596026)
  13. Direct-zol RNA试剂盒(Zymo Research,目录号:R2070)
  14. 高容量cDNA逆转录试剂盒(Thermo Fisher Scientific,Applied Biosystems TM,目录号:4368813)
  15. qPCR引物(表1)

    表1. qPCR引用信息


  16. SYBR Green PCR Master混合物(Thermo Fisher Scientific,Applied Biosystems TM,目录号:4309155)
  17. Hanks的平衡盐溶液(HEPES)(Thermo Fisher Scientific,Gibco TM,目录号:15630080)
  18. 胶原酶II(Sigma-Aldrich,目录号:C1764-50MG)
  19. 牛血清白蛋白(BSA)
  20. 氯化铵(NH 4 Cl)
  21. 碳酸氢钾(KHCO 3 )
  22. 5%EDTA
  23. 叠氮化钠(NaN 3 3)
  24. Iscove修改的Dulbecco's Medium(IMDM)
  25. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号15140122)
  26. 消化缓冲液(参见食谱)
  27. 红细胞裂解缓冲液(见配方)
  28. FACS染色缓冲液(参见食谱)
  29. 完整的培养基(参见食谱)

设备

  1. 移液器
  2. 砂浆和杵
  3. 弯曲剪刀
  4. 新不伦瑞克科学12400培养箱振荡器(Eppendorf,型号:New Brunswick TM 124)
  5. Eppendorf离心机5810R(Eppendorf,型号:5810 R)
  6. TC20自动细胞计数器(Bio-Rad Laboratories,型号:TC20 TM,目录号:1450102)
  7. BD FACSCanto流式细胞分析仪
  8. StepOnePlus实时PCR系统(Thermo Fisher Scientific,Applied Biosystems TM,型号:StepOnePlus TM,目录号:4376600)
  9. DNA引擎Peltier热循环仪(Bio-Rad Laboratories,型号:PTC-200)
  10. NanoDrop 1000分光光度计(Thermo Fisher Scientific,型号:NanoDrop TM 1000)

软件

  1. FlowJo
  2. GraphPad Prism

程序

  1. 从脂肪组织分离基质血管细胞(SVC)(图1)


    图1.从脂肪组织分离基质血管细胞的方案

    1. 18岁至20岁的雄性小鼠按照ICCUAC批准方案进行安乐死。
    2. 用70%乙醇彻底润湿毛皮,然后打开胸腔,收集附睾脂肪组织称重。
    3. 使用弯曲剪刀在100毫米培养皿上煎炸脂肪组织。
    4. 将切碎的组织转移到含有消化缓冲液(含有2%BSA和100mM HEPES的HBSS)的50ml Falcon管中。
    5. 每1g脂肪组织加入10 ml消化缓冲液(参见食谱)。
    6. 在孵化器振荡器(水平固定管)中孵育10分钟,37℃(无组织组织代表完全消化)。
    7. 加入等体积的完整培养基并混合以停止消化。
    8. 通过尼龙活检袋(孔径= 250μm)过滤到新的50ml Falcon管中
    9. 在4℃下以1500×g离心10分钟。
    10. 倾析上清液,并将细胞沉淀重新悬浮于1ml PBS / 2%FBS中
    11. 加入2毫升红细胞裂解缓冲液(参见食谱)(1:2)并混合
    12. 在冰上孵育10分钟。
    13. 加入3毫升完整培养基(1:1)并混合
    14. 以1,000 x g离心5分钟,4°C。
    15. 倾倒上清液。
    16. 将SVC用0.5ml PBS / 2%FBS再悬浮,然后使用TC20自动细胞计数器计数。细胞浓度调整至每毫升2×10 6个细胞,细胞准备进行下游实验。

  2. 流式细胞仪分析
    1. 准备0.5-1×10 6个SVC并重悬于FACS染色缓冲液(参见食谱)。
    2. 按照制造商的指示,用荧光偶联抗体(Materials and Reagents#11)染色SVC。
    3. 在室温下孵育15分钟(光敏;保持在黑暗中)
    4. 加入1ml FACS染色缓冲液
    5. 以1,000×g离心5分钟,4℃。
    6. 倾倒上清液。
    7. 用100-200μlFACS染色缓冲液重新悬浮。
      选项:细胞可以在4℃下在BD TM 稳定固定缓冲液中储存3天,然后用于FACS分析。
    8. 然后样品准备好进行流式细胞分析(图2)或细胞分选仪的特异性免疫细胞分离。


      图2.内脏脂肪组织免疫细胞的流式细胞术分析(VAT)。 将SVC用针对CD11b,F4 / 80,CD11c,CD206,CD19的荧光结合抗体染色,然后通过流式细胞术分析进行检查。 A.脂肪组织巨噬细胞(ATM)定义为CD11b + / F4 / 80 + 亚群,并显示为CD45 + 细胞的百分比。 B.M1和M2 ATM被定义为CD11b F4 / 80 + CD11c + CD206 - 和CD11b 分别为CD206 CD206 + 。这些细胞群体显示为ATM的百分比。 C.B细胞被定义为CD45 CD19 + ,细胞群以CD45 +细胞的百分比表示。

  3. RT-PCR
    1. 将SVC重新悬浮于Trizol中(加入100μlTrizol试剂至<10μg细胞;加入300μlTrizol试剂至<10μg/ ml细胞)中,并在室温(RT)。
    2. 在1000×g离心5分钟(RT)后,将上清液转移到新管中。
    3. 使用Direct-zol RNA试剂盒分离总RNA
    4. RNA浓度用NanoDrop 1000分光光度计测定
    5. 使用高容量cDNA逆转录试剂盒将RNA(500ng)转化为cDNA
    6. 逆转录反应在热循环程序下:25℃10分钟,然后37℃120分钟,然后85℃5分钟,然后4℃。
    7. cDNA在MicroAmp Optical 96孔反应板中是混合引物和SYBR Green PCR Master Mix
    8. 实时PCR(qPCR)反应在热循环条件下运行:95℃10分钟,然后40次循环(95℃15秒,然后60℃1分钟)。
    9. RT-PCR结果显示,贫血SVC表现出比肥胖SVC更高的精氨酸酶1表达,但较少的TNFα丰度(图3A和3B)。


      图3.通过RT-PCR分析和Western印迹的SVC的炎症状态。 精氨酸酶1(A)较少,但更多的TNFα(B )肥胖SVCs的丰度,与瘦SVC相比(qPCR引物信息见表1)。 C.通过蛋白质印迹分析(抗体信息参见材料和试剂#11)评价SVC的NF-κB信号通路的激活(无刺激)。数据以平均值±SEM表示。每组n = 3。 ** 0.01,*** 0.001,Student's t -test。

  4. Western印迹
    参见2011年Zhang的前一篇文章。蛋白质印迹分析表明,肥胖SVC的磷酸化P65水平高于贫血SVC(图3C)。

食谱

  1. 消化缓冲液(新鲜制备),50 ml
    50ml HBSS
    50毫克胶原酶II
    100毫克BSA
    100 mM HEPES
  2. 红血细胞裂解缓冲液(10x),1L
    PBS(不含钙和镁)
    8.3g氯化铵(NH 4 Cl)(150mM)
    1.0g碳酸氢钾(KHCO 3)(10mM)
    1.8ml 5%EDTA(0.1mM)
  3. FACS染色缓冲液,100 ml
    98毫升PBS(不含钙和镁)
    2 ml FBS
    0.1g NaN 3
  4. 完成培养基
    500毫升Iscove修改的Dulbecco的介质(IMDM)
    50ml FBS
    5 ml青霉素 - 链霉素(Thermo Fisher Scientific)

致谢

这个协议是由Ying等人(2017)进行的。本研究由美国心脏病协会(W.PeiT31350039,W. Ying)资助,中国国家自然科学基金(81600610)。

参考

  1. Johnson,AM and Olefsky,JM(2013)。 胰岛素抵抗的起因和驱动因素。 152(4):673-684。
  2. Lumeng,CN,DelProposto,JB,Westcott,DJ和Saltiel,AR(2008)。肥胖的脂肪组织巨噬细胞的表型转换是由巨噬细胞亚型的时空差异产生的。 57(12):3239-3246。
  3. Lumeng,CN,Deyoung,SM,Bodzin,JL和Saltiel,AR(2007)。饮食诱导的肥胖期间招募的脂肪组织巨噬细胞的炎症性质增加。 56(1):16-23。
  4. Nguyen,MT,Favelyukis,S.,Nguyen,AK,Reichart,D.,Scott,PA,Jenn,A.,Liu-Bryan,R.,Glass,CK,Neels,JG和Olefsky,JM(2007) ; 巨噬细胞的亚群渗透肥厚的脂肪组织并被免费激活脂肪酸通过Toll样受体2和4以及JNK依赖性途径。生物化学282(48):35279-35292。
  5. Romeo,GR,Lee,J.和Shoelson,SE(2012)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/22815343”target = “_blank”>代谢综合征,胰岛素抵抗和炎症机制和治疗靶点的作用。 Arterioscler Thromb Vasc Biol 32(8):1771-1776。
  6. Saltiel,AR和Olefsky,JM(2017)。&nbsp; 炎症机制与肥胖和代谢疾病相关联。 J Clin Invest 127(1):1-4。
  7. Weisberg,SP,McCann,D.,Desai,M.,Rosenbaum,M.,Leibel,RL和Ferrante,AW,Jr.(2003)。&lt; a class =“ke-insertfile”href =“http: /www.ncbi.nlm.nih.gov/pubmed/14679176“target =”_ blank“>肥胖与脂肪组织中的巨噬细胞积累相关。 J Clin Invest 112(12): 1796-1808。
  8. Winer,DA,Winer,S.,Shen,L.,Wadia,PP,Yantha,J.,Paltser,G.,Tsui,H.,Wu,P.,Davidson,MG,Alonso,MN,Leong, Glassford,A.,Caimol,M.,Kenkel,JA,Tedder,TF,McLaughlin,T.,Miklos,DB,Dosch,HM和Engleman,EG(2011)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/21499269”target =“_ blank”> B细胞通过调节T细胞和产生致病性IgG抗体来促进胰岛素抵抗。 Nat Med 17(5):610-617。
  9. Ying,W.,Wollam,J.,Ofrecio,JM,Bandyopadhyay,G.,El Ouarrat,D.,Lee,YS,Oh,DY,Li,P.,Osborn,O.and Olefsky,JM(2017)。 &nbsp; 脂肪组织B2细胞通过白三烯LTB4 / LTB4R1促进胰岛素抵抗信号。 J Clin Invest 127(3):1019-1030。
  10. Zhang,H。(2011)。用于检测磷酸化STAT3的Western印迹。 Bio Protoc Bio101:e125。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Vu, J. and Ying, W. (2017). Isolation and Analysis of Stromal Vascular Cells from Visceral Adipose Tissue. Bio-protocol 7(16): e2444. DOI: 10.21769/BioProtoc.2444.
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