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Isolation and Analysis of Stromal Cell Populations from Mouse Lymph Nodes

小鼠淋巴结基质细胞群的分离和分析

YA Yannick O. Alexandre
Scott N Mueller Scott N Mueller

发布: 2017年08月20日第7卷第16期 DOI: 10.21769/BioProtoc.2445 浏览次数: 10425

评审: Ivan ZanoniMeenal SinhaAlesssandro Arduini

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Abstract

Our protocol describes a simple procedure for isolating stromal cells from lymph nodes (LN). LN are disrupted then enzymatically digested with collagenase and dispase to produce a single cell suspension that can be stained with fluorescently labelled antibodies and analysed by flow cytometry. This protocol will enable identification of fibroblastic reticular cells (FRC), lymphatic endothelial cells (LEC), blood endothelial cells (BEC) as PNAd+ BEC that form LN high endothelial venules (HEV). This method can be applied to examine LN stromal cell responses during inflammatory events induced by infections or immunologic adjuvants and to subset most leukocytes found in LN.

Keywords: Lymphoid stromal cells (淋巴基质细胞) search Lymph nodes (淋巴结) search Endothelial cells (内皮细胞) search Fibroblastic reticular cells (成纤维网状细胞) search Immune response (免疫反应) search Virus infection (病毒感染) search

Background

Lymph nodes (LN) are constructed of complex networks of mesenchymal and endothelial stromal cells. These include the fibroblastic reticular cells (FRCs), lymphatic endothelial cells (LECs) and blood endothelial cells (BECs). These stromal cells organize the complex microarchitecture of LN, enabling the support of immune cell migration, homeostasis, tolerance and cellular interactions required for the initiation of immune responses to pathogens and tumors. We have shown that the LN stromal cells can proliferate and expand in response to inflammatory signals and the recruitment of immune cells into LN that accompanies infections (Gregory et al., 2017). These stromal cells can also significantly modulate their transcriptional program to respond to infection, thereby supporting ongoing immune responses. This protocol enables reliable isolation of stromal cell subsets from LN both in the steady state and during disease. This enables phenotypic, functional, genetic or epigenetic investigation of LN stromal cells to reveal how they contribute to tissue homeostasis and immune responses.

Materials and Reagents

  1. 1,000 μl pipette tips (Neptune, catalog number: BT1250 )
  2. 200 μl pipette tips (Neptune, catalog number: BT200 )
  3. 20 μl pipette tips (Neptune, catalog number: BT20 )
  4. 10 μl pipette tips (Neptune, catalog number: BT10XLS3 )
  5. 24-well plate (Corning, catalog number: 3527 )
  6. 10 ml tubes (SARSTEDT, catalog number: 62.9924.284 )
  7. 50 ml tubes (Greiner Bio One International, catalog number: 227261 )
  8. Nylon mesh 70 μm (Clear Edge, catalog number: PA75-1750 )
  9. 5 ml polystyrene round bottom tube (Corning, Falcon®, catalog number: 352008 )
  10. 96-well round-bottom plate (Corning, catalog number: 3788 )
  11. Spatula (VWR, catalog number: 10806-408 )
  12. C57BL\6 mice (THE JACKSON LABORATORY, catalog number: 000664 )
  13. Blank calibration particles (BD, BD Biosciences, catalog number: 556296 )
  14. HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 11344033 )
  15. L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 21051024 )
  16. Benzylpenicillin (CSL, catalog number: AUST R 10329 )
  17. Streptomycin sulphate (Thermo Fisher Scientific, GibcoTM, catalog number: 11860038 )
  18. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  19. RPMI (Thermo Fisher Scientific, GibcoTM, catalog number: 11875093 )
  20. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10099141 )
  21. Collagenase D (Sigma-Aldrich, catalog number: 11088882001 )
    Manufacturer: Roche Diagnostics, catalog number: 11088882001 .
  22. Dispase II (Sigma-Aldrich, catalog number: 04942078001 )
    Manufacturer: Roche Diagnostics, catalog number: 04942078001 .
  23. DNase I (Sigma-Aldrich, catalog number: DN25-1G )
  24. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  25. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906-500G )
  26. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Sigma-Aldrich, catalog number: E5134-1KG )
  27. Antibodies
    1. gp38-PE, 8.1.1 (BioLgend, catalog number: 127408 )
    2. CD31-PE-Cy7, 390 (BioLegend, catalog number: 102524 )
    3. CD45.2-A700, 104 (BioLegend, catalog number: 109822 )
    4. Ter-119-A700, TER-119 (BioLegend, catalog number: 116220 )
    5. HEV-A488, MECA-79 (Thermo Fisher Scientific, eBioscienceTM, catalog number: 53-6036-82 )
    6. CD16/32-purified, 2.4G2 (BD, BD Biosciences, catalog number: 553142 )
  28. Live/Dead Fixable Near-IR cell stain kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: L10119 )
  29. Supplementum Completum (SC) (see Recipes)
  30. RP-2 solution (see Recipes)
  31. Digestion mix 1 (see Recipes)
  32. Digestion mix 2 (see Recipes)
  33. FACS buffer (see Recipes)
  34. Lymph node stromal cell antibody cocktail (see Recipes)

Equipment

  1. Water bath (VWR, catalog number: 89501-464 )
  2. Dumont No.5 forceps (Fine Science Tools, catalog number: 11251-10 )
  3. Graefe forceps (Roboz Surgical Instrument, catalog number: RS-5137 )
  4. Analytical balance for weighing (Ohaus, catalog number: 30208454 )
  5. 1,000 μl pipette (Eppendorf, catalog number: 3121000120 )
  6. 200 μl pipette (Eppendorf, catalog number: 3121000082 )
  7. 20 μl pipette (Eppendorf, catalog number: 3121000040 )
  8. 10 μl pipette (Eppendorf, catalog number: 3121000015 )
  9. Refrigerated centrifuge with plate carriers (Beckman Coulter, model: Allegra® X-15R , catalog number: B09822; Beckman Coulter, catalog number: 368914 )
  10. Flow cytometer (BD, BD Biosciences, model: BD LSRFORTESSA )

Software

  1. FlowJo (FlowJo)
  2. Prism (GraphPad)

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Alexandre, Y. O. and Mueller, S. N. (2017). Isolation and Analysis of Stromal Cell Populations from Mouse Lymph Nodes. Bio-protocol 7(16): e2445. DOI: 10.21769/BioProtoc.2445.

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分类

细胞生物学 > 细胞分离和培养 > 细胞分离

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