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Isolation and in vivo Transfer of Antigen Presenting Cells

抗原递呈细胞的分离和体内转移

PA Pooja Arora
SK Shalu Sharma Kharkwal
SP Steven A. Porcelli

发布: 2014年10月05日第4卷第19期 DOI: 10.21769/BioProtoc.1260 浏览次数: 13650

评审: Jia LiSavita NairAnonymous reviewer(s)

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Abstract

Transfer of antigen presenting cells in vivo is a method used by immunologists to examine the potency of antigen presentation by a selected population of cells. This method is most commonly used to analyze presentation of protein antigens to MHC class I or II restricted T cells, but it can also be used for studies of nonconventional antigens such as CD1-presented lipids. In a recent study focusing on CD1d-restricted glycolipid antigen presentation to Natural Killer T cells, we compared antigen presenting properties of splenic B cells, CD8αPos dendritc cells (DCs) and CD8αNeg DCs (Arora et al., 2014). This protocol describes the detailed method used for isolation of these cell populations, and their transfer into recipient mice to analyze their antigen presenting properties.

As a percentage of total mononuclear cells, an average spleen contains approximately 1-3% myeloid dendritic cells (DCs). In absolute numbers, this translates to approximately 0.6-1.8 x 106 DCs. To enhance the number of DCs in spleen, mice were injected subcutaneously with cells from a cultured melanoma cell line (B16.Flt3L) which has been engineered to express the fms-related tyrosine kinase 3 ligand (Flt3L) (Mach et al., 2000). This protein is a growth factor homologous to colony stimulating factor-1 and plays a critical role in the differentiation of hematopoietic stem cells. Administration of this protein into mice as a purified protein results in the expansion of both CD8αPos and CD8αNeg DC subsets in multiple organs. Similar expansion is also seen in mice that have been implanted with tumor cells overexpressing this protein (Mach et al., 2000). In our experience, up to 60% of the total mononuclear cells in a spleen from a mouse with a palpable B16.Flt3L tumor can be CD11c positive dendritic cells, thereby giving a total yield of up to 5 x 107 DCs per mouse. A schematic illustrating the cell enrichment protocol is included in Figure 1, and representative data on purity of cell populations obtained with this protocol is shown in Figure 2.

Keywords: Isolation (隔离) search Antigen presenting cell (APC) (抗原提呈细胞（APC）) search Dendritic Cell (树突状细胞) search Purification (净化) search Antigen pulsing (Antigen脉冲) search

Materials and Reagents

  1. Source of splenocytes: 6-8 week old female C57BL/6 mice (Jackson ImmunoResearch Laboratories)
  2. Murine B16.Flt3L melanoma cell line [as described by Mach et al. (2000)]
  3. Ultrapure water
  4. 0.05% Trypsin-EDTA (Life Technologies, Gibco®, catalog number: 25300-054 )
  5. Isoflurane (Sigma-Aldrich, catalog number: CDS019936 -250MG)
  6. Collagenase D (Roche Diagnostics, catalog number: 11088858001 )
  7. DNase I, dry powder (QIAGEN, catalog number: 79254 )
  8. 70% Ethanol (prepared from 200 proof ethanol) (Thermo Fisher Scientific, catalog number: 9-6705-004-220 )
  9. RBC lysis buffer (Sigma-Aldrich, catalog number: R7757 )
  10. RPMI-1640 medium with L-glutamine (Life Technologies, Gibco®, catalog number: 11875-119 )
  11. DMEM medium with L-glutamine (Life Technologies, Gibco®, catalog number: 11995-073 )
  12. 200 mM L-glutamine (Life Technologies, catalog number: 25030081 )
  13. MEM non-essential amino acids (Life Technologies, Gibco®, catalog number: 11140-050 )
  14. MEM essential amino acids (Life Technologies, catalog number: 11130-051 )
  15. β-mercaptoethanol (Life Technologies, InvitrogenTM, catalog number: 21985-023 )
  16. Sodium pyruvate (Life Technologies, catalog number: 11360-070 )
  17. HEPES (Life Technologies, InvitrogenTM, catalog number: 15630 )
  18. Phosphate buffered saline (PBS) (Ca2+ and Mg2+ free, pH 7.2) (Life Technologies, InvitrogenTM, catalog number: 20012-050 )
  19. Dulbecco’s PBS (DPBS) with Ca2+ and Mg2+ (Life Technologies, Gibco®, catalog number: 14040-182 )
  20. 0.5 M Ethylenediaminetetraacetate (EDTA) solution (Life Technologies, catalog number: 15575-020 )
  21. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  22. Fetal calf serum (Atlanta Biologicals, catalog number: S11050 )
  23. Penicillin/streptomycin (Life Technologies, InvitrogenTM, catalog number: 15140-163 )
  24. Trypan blue (dry powder) (Sigma-Aldrich, catalog number: T6146 -5G)
  25. Magnetic beads conjugated with anti-mouse CD19 (Miltenyi Biotech, catalog number: 120-000-323 )
  26. Magnetic beads conjugated with anti-mouse CD11c (Miltenyi Biotech, catalog number: 130-152-001 )
  27. CD8αPos mouse DC isolation kit (Miltenyi Biotech, catalog number: 130-091-169 )
  28. Fc-gamma receptor blocking antibody (Clone 2.4G2) (BD Biosciences, catalog number: 553141 ).
  29. Anti-mouse CD11c-FITC (BD Biosciences, catalog number: 553801 )
  30. Anti-mouse CD8α-PerCP (BD Biosciences, catalog number: 553036 )
  31. Anti-mouse B220-PE (BD Biosciences, catalog number: 553090 )
  32. 0.08% trypan blue (see Recipes)
  33. Serum free DMEM and RPMI media (see Recipes)
  34. Complete RPMI and DMEM media (see Recipes)
  35. MACS buffer (see Recipes)
  36. FACS staining buffer (see Recipes)
  37. 10x collagenase D solution (see Recipes)

Equipment

  1. 1 ml syringes (BD, catalog number: 26048 )
  2. 23 G1 needle (BD, catalog number: 305145 )
  3. 100 mm Petri dishes (Thermo Fisher Scientific, catalog number: 0875712 )
  4. Surgical instruments (Kent Scientific, catalog number: INSMOUSEKIT )
  5. Cell strainer (70 µm) (BD, catalog number: 352350 )
  6. Large Petri plates (Thermo Fisher Scientific, catalog number: FB0875712 )
  7. Vacuum filtration system (500 ml, 0.22 um) (Corning, catalog number: 431097 )
  8. LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
  9. Magnetic stand MACS separator (Miltenyi Biotec, catalog number: 130-042-302 )
  10. Wide-bore 200 μl pipette tips (PerkinElmer, catalog number: 111623 )
  11. Corning ultra-low attachment 96-well plates (Corning, catalog number: CLS3474-24EA )

Procedure

English
中文翻译

文章信息

版权信息

© 2014 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Arora, P., Kharkwal, S. S. and Porcelli, S. A. (2014). Isolation and in vivo Transfer of Antigen Presenting Cells. Bio-protocol 4(19): e1260. DOI: 10.21769/BioProtoc.1260.

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分类

免疫学 > 免疫细胞功能 > 抗原特异反应

免疫学 > 免疫细胞功能 > 淋巴细胞

免疫学 > 免疫细胞分离 > 淋巴细胞

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