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Process and Analysis of Kidney Infiltrates by Flow Cytometry from Murine Lupus Nephritis
从狼疮性肾炎小鼠模型中分离肾脏炎症细胞及流式细胞术分析   

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Abstract

Methods for the isolation and characterization of mononuclear phagocytes from the kidneys of mice with SLE are essential to understand the patho-physiology of the disease. Activation of these cells is associated with the onset of clinical disease in mice and infiltration with these cells is associated with poor prognosis in humans.An analysis of the function of these cells should lead to a better understanding of the inflammatory processes that lead to renal impairment in SLE and other renal inflammatory diseases.

Keywords: SLE nephritis(SLE肾炎), Kidney infiltrates(肾infiltrates), Macrophages from kidney(肾macrophages from), Collagen digestion(胶原消化), Inflammation(炎症)

Materials and Reagents

  1. Fetal bovine serum (FBS)
  2. Sterile PBS (Life Technologies, Invitrogen™, catalog number: 20012-027 )
  3. 0.17M Ammonium chloride
  4. Collagenase Type I (CLS I) (Worthington, catalog number: 4197 , specific activity 230 U mg-1)
  5. DMEM, High glucose (Life Technologies, Gibco®, catalog number: 10313 )
  6. Paraformaldehyde (Tousimis, catalog number: 1108A )
  7. BSA (Fraction V) (Sigma-Aldrich, catalog number: A7030 )
  8. Fc block (CD16/CD32)
  9. FACS staining buffer (see Recipes)

Equipment

  1. BD LSRII or similar flow cytometer
  2. Bench-top refrigerated centrifuge
  3. BD cell strainer (40 nm) (BD Biosciences, Falcon®, catalog number: 352340 )
  4. 30 ml syringe (BD Biosciences, Falcon®, catalog number: 309661 )
  5. Microscopes
  6. 21G Needles (BD Biosciences, Falcon®, catalog number: 305165 )
  7. 26G needles (BD Biosciences, Falcon®, catalog number: 305111 )
  8. V bottom 96 well Assay plate (Corning, Costar® , catalog number: 3897 )
  9. Glass slides Frosted (Thermo Fisher Scientific, catalog number: 12-550-11 )

Procedure

  1. Procedure for harvesting the kidney from nephritic mice for analysis of kidney infiltrates.
    1. Anesthetize the mouse and perfuse with 60 ml of cold PBS over 3-5 min through the left ventricle after snipping the right atrium, and observe for pale white color change in liver and kidney. If needed, repeat perfusion with another 60 ml of cold PBS.
    2. Carefully remove and cut the kidneys into 1 to 2 mm3 pieces, excluding any adjoining renal fat.
    3. Incubate the slices in DMEM containing 2 mg/ml Collagenase Type I (Worthington) for 30 min at 37 °C (use 10 ml per two kidneys).
    4. Gently disrupt the tissue by pipetting up and down sequentially through 25 ml, 10 ml, and 5 ml pipettes to obtain a fine cell suspension.
    5. Filter the cell suspension through a BD cell strainer (70 nm) into a conical tube.
    6. Gently rub the remaining material between two glass slides, resuspend in 2 ml DMEM, filter and add to the suspension.
    7. Allow the suspension to settle briefly (3-5 min) during which most of the larger fragments settle to the bottom. Harvest the suspension excluding the bottom 200 μl containing the fragments.
    8. Examine the settled cells under a microscope to see if any clumps are present. If so, resuspend the settled cells in fresh DMEM, filter and repeat step 6.
    9. Pool the suspension(s) obtained and centrifuge at 1,200 rpm for 10 min.
    10. Decant supernatant; resuspend the pellet in 5 ml of ice cold ammonium chloride (0.17 M, pH 7.2) for 5 min on ice.
    11. Add 15 ml of serum free DMEM. Count cells to estimate the number of total cells in the suspension. Spin at 1,200 rpm for 5 min.
    12. Resuspend cells in 1 ml of FACS buffer (3% fetal calf serum in PBS). Cells are now ready for flow cytometric analysis or further isolation procedures.

  2. Procedure for the analysis of kidney infiltrates by flow cytometry
    1. Reuspend the kidney cells in Fc block (CD16/CD32) for 15 min, adjusting the total suspension volume to approximately 100 µl per stain. Use a maximum of 1 ml (10 stains) for one whole kidney.
    2. Distribute the cells accordingly into a 96 well V bottom plate (100 µl per well).
    3. Add biotinylated antibodies (1/200 dilution), mix gently and incubate for 30 min on ice. Keep the plates in the dark throughout the incubation.
    4. Meanwhile, mix the next cocktail of antibodies of your choice together with the streptavidin fluorochrome in a single tube (1/200 dilution of each antibody in FACS staining buffer). Include a set of stains using isotype controls.
    5. To remove the unbound or excess stain add 100 μl of ice cold PBS to the wells after 30 min of incubation. Centrifuge the plate for 5 min at 1,200 rpm at 4 °C.
    6. Discard the liquid by inverting the plate once without disturbing the cell pellet.
    7. Add the cocktail of antibodies from step 4 to the pellet (100 µl /well) and gently resuspend using a multichannel pipette. Incubate the plate for another 30 min on ice.
    8. Repeat steps 5 and 6.
    9. Resuspend the cells in 200 µl of 2% paraformaldehyde, transfer and store in FACS tubes in the dark until the time of acquisition on the flow cytometer (within 24 h).
    10. Analysis of the acquired cells was carried out in flowjo software to identify the type of cells within the infiltrates and their percentage within the kidney.

Notes

Kidneys need to be perfused with PBS to remove blood before processing since large numbers of CD11b+ cells appear in the blood in SLE models. Perfusion should begin with the heart still beating to improve circulation of the PBS. If the liver is uniformly pale after perfusion then blood removal has been adequate. Blood removal also improves the quality of immunohistochemistry.

Recipes

  1. 0.17 M ammonium chloride
  2. FACS staining buffer
    PBS
    3% FBS

Acknowledgments

This work was supported by the NY SLE foundation to RB and National Institutes of Health RO1 DK085241-01 to AD.

References

  1. Bethunaickan, R., Berthier, C. C., Ramanujam, M., Sahu, R., Zhang, W., Sun, Y., Bottinger, E. P., Ivashkiv, L., Kretzler, M. and Davidson, A. (2011). A unique hybrid renal mononuclear phagocyte activation phenotype in murine systemic lupus erythematosus nephritis. J Immunol 186(8): 4994-5003.

简介

从SLE小鼠的肾中分离和表征单核吞噬细胞的方法对于了解疾病的病理生理学是必不可少的。 这些细胞的活化与小鼠中的临床疾病的发作相关,并且这些细胞的浸润与人类的不良预后相关。这些细胞的功能的分析应当导致更好地理解导致肾损伤的炎症过程 在SLE等肾性炎症性疾病中的应用。

关键字:SLE肾炎, 肾infiltrates, 肾macrophages from, 胶原消化, 炎症

材料和试剂

  1. 胎牛血清(FBS)
  2. 无菌PBS(Life Technologies,Invitrogen TM,目录号:20012-027)
  3. 0.17M氯化铵
  4. 胶原酶I型(CLS I)(Worthington,目录号:4197,比活性230U mg -1
  5. DMEM,高葡萄糖(Life Technologies,Gibco ,目录号:10313)
  6. 多聚甲醛(Tousimis,目录号:1108A)
  7. BSA(级分V)(Sigma-Aldrich,目录号:A7030)
  8. Fc区(CD16/CD32)
  9. FACS染色缓冲液(参见配方)

设备

  1. BD LSRII或类似的流式细胞仪
  2. 台式冷冻离心机
  3. BD细胞过滤器(40nm)(BD Biosciences,Falcon ,目录号:352340)
  4. 30ml注射器(BD Biosciences,Falcon ,目录号:309661)
  5. 显微镜
  6. 21G针(BD Biosciences,Falcon ,目录号:305165)
  7. 26G针(BD Biosciences,Falcon ,目录号:305111)
  8. V底96孔测定板(Corning,Costar ,目录号:3897)
  9. 玻璃玻璃磨砂(Thermo Fisher Scientific,目录号:12-550-11)

程序

  1. 从肾炎小鼠收获肾脏以分析肾脏浸润的程序。
    1. 麻醉鼠标和灌注60毫升冷PBS超过3-5分钟通过左心室切断右心房后,观察肝脏和肾脏的苍白色变化。 如果需要,用另外60ml冷PBS重复灌注。
    2. 小心地将肾脏切除并切成1至2毫米 3片,不包括任何相邻的肾脏脂肪。
    3. 将切片在含有2mg/ml I型胶原酶(Worthington)的DMEM中在37℃下孵育30分钟(使用每两个肾脏10ml)。
    4. 轻轻破碎组织通过吸取上下顺序通过25毫升,10毫升和5毫升移液器获得细胞悬浮液。
    5. 将细胞悬浮液通过BD细胞过滤器(70nm)过滤到锥形管中
    6. 轻轻擦拭两个载玻片之间的剩余材料,重悬于2ml DMEM中,过滤并加入悬浮液中。
    7. 让悬浮液短暂停留(3-5分钟),在此期间大多数较大的碎片沉降到底部。收获包含碎片的底部200μl以外的悬浮液。
    8. 在显微镜下检查沉降的细胞,看是否有任何团块。如果是,将沉降的细胞重悬于新鲜DMEM中,过滤并重复步骤6
    9. 收集获得的悬浮液,并以1,200rpm离心10分钟
    10. 倾析上清液; 在冰上将沉淀重悬于5ml冰冷的氯化铵(0.17M,pH7.2)中5分钟。
    11. 加入15ml无血清DMEM。 计数细胞以估计悬浮液中总细胞的数量。 以1,200rpm旋转5分钟。
    12. 重悬细胞在1毫升FACS缓冲液(3%胎牛血清的PBS溶液)。 细胞现在可以进行流式细胞分析或进一步的分离程序
  2. 通过流式细胞术分析肾浸润的程序
    1. 将Fc细胞(CD16/CD32)中的肾脏细胞再悬浮15分钟,将总悬浮液体积调整至约100μl/染色。对于一个全肾最多使用1ml(10个染色剂)。
    2. 将细胞相应地分配到96孔V底板(每孔100μl)
    3. 加入生物素化的抗体(1/200稀释),轻轻混合并在冰上孵育30分钟。在孵化过程中保持板在黑暗中。
    4. 同时,将单一管中的所选抗体的下一个混合物与链霉亲和素荧光染料混合(在FACS染色缓冲液中每种抗体的1/200稀释度)。使用同种型控件包括一组污渍。
    5. 为了去除未结合或过量的染色,在温育30分钟后向孔中加入100μl冰冷的PBS。在4℃下以1,200rpm离心板5分钟
    6. 通过颠倒板一次而不干扰细胞沉淀来弃去液体。
    7. 将步骤4的抗体的混合物加入沉淀(100μl/孔),并使用多通道移液管轻轻地重悬。 在冰上再孵育30分钟。
    8. 重复步骤5和步骤6.
    9. 重悬细胞在200微升2%多聚甲醛,转移和存储在FACS管在黑暗中直到流式细胞仪上获取的时间(24小时内)。
    10. 在flowjo软件中对获得的细胞进行分析以鉴定浸润物内的细胞类型及其在肾脏内的百分比。

笔记

肾脏需要用PBS灌注以在处理前除去血液,因为在SLE模型中大量的CD11 b + 细胞出现在血液中。 灌注应该从心脏仍然跳动开始,以改善PBS的循环。 如果肝脏在灌注后均匀地变苍白,那么除血已经足够了。 血液去除也提高了免疫组化的质量。

食谱

  1. 0.17M氯化铵
  2. FACS染色缓冲液
    PBS
    3%FBS

致谢

这项工作是由NY SLE基金会RB和国家卫生研究院RO1 DK085241-01到AD支持。

参考文献

  1. Bethunaickan,R.,Berthier,CC,Ramanujam,M.,Sahu,R.,Zhang,W.,Sun,Y.,Bottinger,EP,Ivashkiv,L.,Kretzler,M.and Davidson, 。 鼠系统性红斑狼疮肾炎中的独特杂合性肾单核吞噬细胞活化表型。 J Immunol 186(8):4994-5003。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bethunaickan, R. and Davidson, A. . (2012). Process and Analysis of Kidney Infiltrates by Flow Cytometry from Murine Lupus Nephritis. Bio-protocol 2(9): e167. DOI: 10.21769/BioProtoc.167.
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