Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

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: 17806

评审: Kathrin SutterAndrea IntroiniAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Nature Immunology, featuring study using the protocol.

Feb 2014

Advertisement

EasySep 免疫磁珠细胞分选
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

饮食诱导性肥胖模型中脂肪组织炎症性与代谢激活型巨噬细胞的鉴定与分选

饮食诱导性肥胖模型中脂肪组织炎症性与代谢激活型巨噬细胞的鉴定与分选

Dan Wu [...] Weidong Wang

2025年10月20日 2117 阅读

基于 PKD2L1 启动子驱动的慢病毒系统在体外选择性富集与鉴定脑脊液接触神经元

基于 PKD2L1 启动子驱动的慢病毒系统在体外选择性富集与鉴定脑脊液接触神经元

Wei Tan [...] Qing Li

2025年11月20日 1259 阅读

重新优化原代小胶质细胞分离流程：改进的小胶质细胞提取方法

重新优化原代小胶质细胞分离流程：改进的小胶质细胞提取方法

Jianwei Li [...] Guohui Lu

2025年12月05日 1143 阅读

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.

ris ris Download Citation in RIS Format

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

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件