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Peer-reviewed

In vitro Differentiation of Murine Innate Lymphoid Cells from Common Lymphoid Progenitor Cells

基于鼠源淋巴祖细胞的体外分化获得先天免疫性淋巴细胞的方法

CS Corey Seehus
JK Jonathan Kaye

发布: 2016年03月20日第6卷第6期 DOI: 10.21769/BioProtoc.1770 浏览次数: 9670

评审: Ivan ZanoniKathrin SutterMarielle Cavrois

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The authors used this protocol in:

Cover of Nature Immunology, featuring study using the protocol.

May 2015

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Abstract

Subtypes of innate lymphoid cells (ILC), defined based on their cytokine secretion profiles and transcription factor expression, are important for host protection from pathogens and maintaining tissue homeostasis. ILCs develop from common lymphoid progenitors (CLP) in the bone marrow. Using the methods described here, we have previously shown that loss of the transcriptional regulator TOX (Thymocyte-selection associated HMG-box protein) leads to specific changes in ILC development and differentiation. Here, we describe how to obtain ILCs from in vivo isolated CLP grown in vitro.

Materials and Reagents

  1. 5 ml Syringe (BD biosciences, catalog number: 309646 )
  2. 25 G x 5/8 Needle (BD biosciences, catalog number: 305122 )
  3. 15 ml Conical Tubes (Corning, Falcon, catalog number: 352097 )
  4. 50 ml Conical Tubes (USA Scientific, catalog number: 1500-1811 )
  5. 60 x 15 mm culture dish (Corning, Falcon, catalog number: 351007 )
  6. Sterile 48 well plates, tissue culture treated (Greiner Bio-One GmbH, catalog number: 677180 )
  7. 5 ml and 10 ml serological pipets
  8. OP9-DL1 cells (not commercially available, must privately request from Dr. J. C. Zuniga-Pflucker, University of Toronto, Sunnybrook Research Institute)
  9. OP9-DL1 stromal cells (OP9-DL1 made by and obtained from Dr. J. C. Zuniga-Pflucker)
  10. Antibodies (all purchased from eBiosciences, Affymetrix)
    1. Blocking antibody Anti-CD16/CD32 Functional Grade Purified (clone 93) (eBioscience, Affymetrix, catalog number: 16-0161 )
    2. Anti-mouse lineage antibodies (concentrations as per vendor instructions), Fluorochromes were conjugated to FITC or APC (Lineage markers), APC-Cy7 (IL7Rα), PerCP eFluor® 710 (Flt-3) and PE-Cy7 (Ly6A/E)
      CD4 (clone 6K1.5)
      CD8α (53-6.7)
      CD3ε (145-2C11)
      CD11b (M1/70)
      CD11c (N418)
      CD19 (1D3)
      B220 (RA3-6B2)
      Gr-1 (RB6-8C5)
      Nk1.1 (PK136)
      Ter-119 (TER-119)
      Thy1.2 (30-H12)
    3. Common lymphoid progenitor defining cell surface antibodies (concentrations as per vendor instructions)
      Flt3 (A2F10)
      IL7Rα (A7R34)
      Ly6A/E (Sca-1) (D7)
  11. Dulbecco’s Phosphate Buffered Saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-144 )
  12. Dulbecco’s Modified Eagle Medium, High Glucose (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11965-092 )
  13. Minimum Essential Media (MEM) alpha, No Nucleosides (Thermo Fisher Scientific, GibcoTM, catalog number: 12561-049 )
  14. Fetal Bovine Serum (FBS) (Omega Scientific, catalog number: FB-01 )
  15. 100x Penicillin-Streptomycin (PSA) (Thermo Fisher Scientific, GibcoTM, catalog number: 10378-016 )
  16. Sodium Azide (Sigma-Aldrich, catalog number: S2002 )
  17. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M7522 )
  18. Sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360-070 )
  19. MEM nonessential amino acids (Mediatech, Corning® glutagroTM, catalog number: 25-025-CI )
  20. HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630 )
  21. Trypsin-EDTA (0.05%), phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 25300-054 )
  22. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A8022 )
  23. Recombinant mouse Interleukin 7 (IL-7) carrier free (Biolegend, catalog number: 577802 )
  24. Recombinant mouse Stem Cell Factor (SCF) carrier free (Biolegend, catalog number: 579702 )
  25. Recombinant mouse IL-33 carrier free (Biolegend, catalog number: 580504 )
  26. Mitomycin C (Santa Cruz Biotechnology, catalog number: sc-3514 )
  27. Phosphate buffered saline (PBS)
  28. NaN3
  29. FACS Buffer (see Recipes)
  30. Sterile Sorting Buffer (see Recipes)
  31. Innate lymphoid cell media (ILC media) (see Recipes)
  32. OP9-DL1 media (see Recipes)

Equipment

  1. Sterile Biosafety cabinet
  2. Incubator set at 37 °C, 5% CO2
  3. Vortex Genie 2 (Thermo Fisher Scientific, catalog number: 12-812 )
  4. Sterile FACS Tubes
  5. Cell sorter (BD Biosciences, model: FACS aria III )
  6. Reichert Bright-Line Hemocytometer (Sigma-Aldrich, catalog number: Z359629 )
  7. Dissection scissors (Roboz Surgical Instrument Co., catalog number: RS-5912 )
  8. Forceps (Roboz Surgical Instrument Co., catalog number: RS-5137 )

Procedure

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文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Seehus, C. and Kaye, J. (2016). In vitro Differentiation of Murine Innate Lymphoid Cells from Common Lymphoid Progenitor Cells. Bio-protocol 6(6): e1770. DOI: 10.21769/BioProtoc.1770.

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分类

免疫学 > 免疫细胞分离 > 维持和分化

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