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Isolation of Dendritic Cells and Macrophages from the Murine Kidneys of Lupus by Cell Sorter

流式细胞术分离狼疮小鼠肾脏的树突状细胞和巨噬细胞

Ramalingam Bethunaickan Ramalingam Bethunaickan
AD Anne Davidson

发布: 2012年04月20日第2卷第8期 DOI: 10.21769/BioProtoc.168 浏览次数: 25534

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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.

Background

Materials and Reagents

  1. Fetal bovine serum (FBS)
  2. Sterile PBS (Life Technologies, Invitrogen™, catalog number: 20012-027 )
  3. 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: A-7030 )
  8. FACS antibodies:
    1. FC block (BD Biosciences, Falcon®, catalog number: 553142 )
    2. CD11B APC (BD Biosciences, Falcon®, catalog number: 553310 )
    3. F/480 FITC  (Serotech Laboratories, catalog number: MCA497FB )
    4. Streptavidin PERCP (BD Biosciences, Falcon®, catalog number: 554064 )
    5. CD4, PE (BD Biosciences, Falcon®, catalog number: 553049 )
    6. CD5, PE (eBioscience, catalog number: 12-0051-82 )
    7. B220, PE (BD Biosciences, Falcon®, catalog number: 553090 )
    8. CD49b, PE (BD Biosciences, Falcon®, catalog number: 558759 )
    9. CD11C (Biotin, catalog number: 553800 )
    10. Streptavidin PERCP (BD Biosciences, Falcon®, catalog number: 554064)
  9. DAPI (MP Biomedicals, catalog number: 157574 )
  10. FACS staining buffer (see Recipes)

Equipment

  1. FACS Aria or similar cell sorter
  2. Bench-top refrigerated centrifuge
  3. BD cell strainer (40 nm) (BD Biosciences, Falcon®, catalog number: 352340 )
  4. Conical tube
  5. 30 ml syringe (BD Biosciences, Falcon®, catalog number: 309661 )
  6. 21G Needles (BD Biosciences, catalog number: 305165 )
  7. 26G needles (BD Biosciences, 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

English
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文章信息

版权信息

© 2012 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Bethunaickan, R. and Davidson, A. (2012). Isolation of Dendritic Cells and Macrophages from the Murine Kidneys of Lupus by Cell Sorter. Bio-protocol 2(8): e168. DOI: 10.21769/BioProtoc.168.

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分类

免疫学 > 免疫细胞分离 > 巨噬细胞

细胞生物学 > 基于细胞的分析方法 > 流式细胞术

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