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Isolation of Splenic Dendritic Cells Using Fluorescence-activated Cell Sorting

采用流式细胞术分离脾脏中树突细胞

ST Simon J Tavernier
FO Fabiola Osorio
SJ Sophie Janssens
Bart N Lambrecht Bart N Lambrecht

发布: 2015年03月05日第5卷第5期 DOI: 10.21769/BioProtoc.1415 浏览次数: 17294

评审: Kathrin SutterAndrea IntroiniAnonymous reviewer(s)

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Abstract

The spleen is a vastly vasculated organ and consists of a complex organized network of innate and adaptive immune cells. This permits the specialized functions of the spleen such as antibacterial and antifungal immunity and iron metabolism among others (Mebius and Kraal, 2005). Different dendritic cell (DC) subsets reside in the spleen and can be defined by the expression of unique surface markers. These DC subsets are recognized to perform non-redundant functions in the immune system (Merad et al., 2013). In our recent study, we found that Inositol Requiring Enzyme (IRE)-1 is specifically activated in splenic CD8a+ DCs. Furthermore, loss of X-box binding protein (XBP)-1 – the transcription factor regulated by IRE-1 – resulted in defective cross-presentation of dead cell associated antigens by splenic CD8a+ DCs (Osorio et al., 2014). This protocol allows the isolation of specific DC subsets for experimental use ex-vivo.

Materials and Reagents

  1. Mice (Jackson Laboratories, C57Bl/6 )
  2. 2,2,2-tribromoethanol (Sigma-Aldrich, catalog number: T48402 )
  3. 2-methyl-2-butanol (Sigma-Aldrich, catalog number: 240486 )
  4. PBS (Gibco, catalog number: 10010-015 )
  5. 0.5 M EDTA (Lonza, catalog number: 51234 )
  6. 2-mercaptoethanol (Sigma-Aldrich, catalog number M3148 )
  7. BSA (Amresco, catalog number: 0332-500g )
  8. HBSS (Life Technologies, catalog number: 24020-091 )
  9. RPMI 1640 GlutaMax (Life Technologies, catalog number: 61870-010 )
  10. Gentamycin (Gibco, catalog number: 15710-049 )
  11. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F7524 )
  12. Liberase TM (Roche Diagnostics, catalog number: 0 5401127001 ) (dilute 50 mg in 50 ml of RPMI, aliquot and stored at -20 °C)
  13. DNase I (Roche, catalog number: 0 4536282001 ) (dilute content in 1 ml PBS, aliquot and stored at -20 °C)
  14. FACS antibodies
    1. CD3 - FITC (eBioscience, clone 145-2C11, dilution 1/300)
    2. CD19 - FITC (eBioscience, clone 1D3, dilution 1/500)
    3. CD11c - PE-eFluor610 (eBioscience, N418, dilution 1/300)
    4. MHCII - APC-Cy7 (Biolegend, clone M5/114.15.2, dilution 1/1,000)
    5. CD8a - PE-Cy5 (BD-Pharmingen, clone 53-6.7, dilution 1/600)
    6. CD11b - PE-Cy7 (BD-Pharmingen, clone M1/70, dilution 1/800)
    7. CD64 - AF647 (BD-Pharmingen, clone X54-5/7.1, dilution 1/100)
    8. CD16/CD32 (Produced in-house, clone 2.4G2, dilution 1/200)
      Note: The use of 2.4G2 minimizes background fluorescence by inhibiting non-specific binding of antibodies to FcG – receptors present on immune cells.
  15. Fixable viability dye eFluor 506 (eBioscience, catalog number: 65-0866-18 )
    1. Reconstitute vial with 400 μl PBS
    2. Aliquot and store vials at -80 °C
    3. Use at a dilution of 1/200
  16. Anti-FITC microbeads (Miltenyi, catalog number 130-048-701 )
  17. LD columns (Miltenyi, catalog number: 130-042-901 )
  18. Ultra comp eBeads (eBioscience, catalog number: 01-2222-42 )
  19. Trypan blue (Sigma-Aldrich, catalog number: T8154 )
  20. Avertin (see Recipes)
  21. 2x digestion medium (see Recipes)
  22. MACS buffer (see Recipes)
  23. R10 medium (see Recipes)
  24. Osmotic lysis buffer (see Recipes)

Equipment

  1. 100 μm Nylon cell strainer (Falcon, catalog number: 352360 )
  2. 15 ml tubes (Falcon, catalog number: 430791 )
  3. 35 mm Petri Dishes
  4. 5ml syringe (Norm-Ject, catalog number: 4020-000V0 )
  5. 25 Gauge needle (Terumo, catalog number: NN-2516R )
  6. Pasteur pipette
  7. Forceps
  8. Surgical scissor/scalpel
  9. 37 °C warm water bath
  10. Shaker
  11. Centrifuge
  12. Aspiration device
  13. MACS MultiStand (Miltenyi, catalog number: 130-042-303 )
  14. Midi MACS (Miltenyi, catalog number: 130-042-302 )
  15. Polystyrene 5 ml tubes with 100 μm cell strainer cap (BD Falcon, catalog number: 352235 )
  16. Polypropylene 5ml tubes (BD Falcon, catalog number: 352063 )
  17. Microscope
  18. Bürker-Türk counting plate
  19. Multicolour FACS sorter (BD Aria II or equivalent cell sorter equipped with 405 nm, 488 nm and 633 nm lasers and appropriate filterset)

Procedure

English
中文翻译

文章信息

版权信息

© 2015 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Tavernier, S. J., Osorio, F., Janssens, S. and Lambrecht, B. N. (2015). Isolation of Splenic Dendritic Cells Using Fluorescence-activated Cell Sorting. Bio-protocol 5(5): e1415. DOI: 10.21769/BioProtoc.1415.

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分类

免疫学 > 免疫细胞分离 > 抗原递呈细胞

免疫学 > 免疫细胞染色 > 流式细胞术

细胞生物学 > 细胞分离和培养 > 细胞分离

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