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Adoptive Transfer of Lung Antigen Presenting Cells

肺抗原呈递细胞的过继转移

XZ Xiaofeng Zhou
BM Bethany B Moore

发布: 2017年03月20日第7卷第6期 DOI: 10.21769/BioProtoc.2182 浏览次数: 10541

评审: Ivan ZanoniKathrin SutterAnonymous reviewer(s)

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Cover of Mucosal Immunology, featuring study using the protocol.

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Abstract

Our protocol describes adoptive transfer of antigen presenting cells (APCs) isolated from the lungs by enzymatic digestion and magnetic enrichment. This protocol can be used to study APC functions and trafficking.

Keywords: Lung antigen presenting cells (肺抗原呈递细胞) search Dendritic cells (树突状细胞) search Macrophages (巨噬细胞) search Adoptive transfer (过继转移) search Enzymatic digestion (酶消化) search Magnetic cell isolation (磁性细胞分离) search Intravenous injection (静脉注射) search

Background

Lung APCs, including macrophages and dendritic cells (DCs), play a critical role in sensing invading pathogens, priming T cell responses and controlling tolerogenic responses. Lung DCs are the most potent professional APCs, including conventional DCs (cDCs) and plasmacytoid DCs (pDCs) during steady-state, and newly recruited monocyte-derived DCs (moDCs) upon inflammation (Kopf et al., 2015). Lung resident macrophages, including alveolar macrophages, interstitial macrophages and bronchial macrophages, are less potent in presenting antigens.

All macrophages and cDCs express high levels of CD11c on their cell surface, while pDCs express intermediate levels of CD11c (Becher et al., 2014). Thus, CD11c magnetic microbeads can be used to isolate mouse lung macrophages and cDCs. We developed a protocol of isolating lung APCs from normal mice and then adoptively transferring them to syngeneic bone marrow transplanted mice. We used this protocol to determine whether normal lung APCs are sufficient to restore T helper cell polarization in response to herpesvirus infection post-transplant (Zhou et al., 2016).

Materials and Reagents

  1. PrecisionGlide needles 23 G (BD, catalog number: 305111 )
  2. PrecisionGlide 26 G (BD, catalog number: 305193 )
  3. 60 x 15 mm Petri dish (Corning, Falcon®, catalog number: 351007 )
  4. 50 ml flip-top conical centrifuge tubes (Thermo Fisher Scientific, catalog number: 362696 )
  5. 10 ml Luer slip tip syringe (Fisher Scientific, catalog number: 14-841-55 )
  6. Razor blade
  7. Tissue culture plates
  8. 100 µm nylon screen (Nylon Mesh Lab Pack) (Sefar, catalog number: 7050-1220-000-20 )
  9. MACS LS column (Miltenyi Biotec, catalog number: 130-042-401 )
  10. 15 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352097 )
  11. 1 ml slip tip syringe (BD, catalog number: 309659 )
  12. 0.22 µm filter (Millipore Express PLUS 0.22 µm PES) (EMD Millipore, catalog number: SCGPU01RE )
  13. Pipette tips 
  14. Latex gloves
  15. Syringe tip caps (BD, catalog number: 305819 )
  16. Mice
  17. 70% ethanol
  18. Phosphate buffered saline (PBS, pH 7.4) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  19. Heat-inactivated fetal bovine serum (Mediatech, catalog number: 35-010-CV )
  20. Penicillin-streptomycin 100x (Mediatech, catalog number: 30-002-CI )
  21. L-glutamine 100x, 200 mM (Thermo Fisher Scientific, GibcoTM, 25030-018 )
  22. Amphotericin B (Thermo Fisher Scientific, GibcoTM, catalog number: 15290018 )
  23. Dulbecco’s modified Eagle’s medium (DMEM) (Lonza, catalog number: 12-604F )
  24. Collagenase A (Roche Diagnostics, catalog number: 10103578001 )
  25. DNase I (Sigma-Aldrich, catalog number: D4263 )
  26. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: 254134 )
  27. Potassium bicarbonate (KHCO3) (Sigma-Aldrich, catalog number: 431583 )
  28. EDTA, 0.5 M (Lonza, catalog number: 51201 )
  29. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3912 )
  30. Percoll (Sigma-Aldrich, catalog number: P1644 )
  31. Mouse CD11c MicroBeads UltraPure (Miltenyi Biotec, catalog number: 130-108-338 )
  32. Trypan blue solution, 0.4% (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  33. Complete media (see Recipes)
  34. Collagenase digest solution (see Recipes)
  35. RBC lysis buffer (see Recipes)
  36. Serum free media (see Recipes)
  37. MACS buffer (see Recipes)

Equipment

  1. CO2 tank
  2. Scissors
  3. Forceps
  4. Water bath
  5. Cell culture incubator
  6. Rocker
  7. Refrigerated Swing bucket centrifuge (Beckman Coulter, model: Allegra X-12R )
  8. Vortex
  9. Hemocytometer
  10. Mouse restrainer
  11. Heat lamp
  12. Pipette 
  13. Compound microscope
  14. MACS separator (Miltenyi Biotec)

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

Zhou, X. and Moore, B. B. (2017). Adoptive Transfer of Lung Antigen Presenting Cells. Bio-protocol 7(6): e2182. DOI: 10.21769/BioProtoc.2182.

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分类

免疫学 > 免疫细胞功能 > 巨噬细胞

细胞生物学 > 细胞移植 > 免疫细胞过继转移

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