Assessment of Human Dendritic Cell Antigen Uptake by Flow Cytometry
采用流式细胞术评估人树突细胞的抗原摄取
发布: 2013年11月20日第3卷第22期 DOI: 10.21769/BioProtoc.974 浏览次数: 17696
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Mar 2013
Abstract
Antigen uptake by dendritic cells is the first key step towards induction of antigen-specific T-cell responses. This flow cytometry-based protocol describes the analysis of dendritic cell uptake of soluble antigens through two different mechanisms: non-specific macropinocytosis (using Lucifer Yelloy CH), and receptor-mediated endocytosis (using DQTM Ovalbumin). The protocol is generated based on data presented in Olivar et al. (2013).
Keywords: Dendritic cells (树突状细胞)
Endocytosis (内吞作用)
Flow cytometry (流式细胞仪)
Fluorescent dye (荧光染料)
Materials and Reagents
- Whole blood
- RPMI 1640 Medium, GlutaMAXTM (Gibco®, catalog number: 61870 )
- DPBS without Ca2+ and Mg2+ (Gibco®, catalog number: 14190-169 )
- 100x liquid Penicillin-Streptomycin (Gibco®, catalog number: 15140-122 )
- 200 mM L-Glutamine solution (Gibco®, catalog number: 25030-024 )
- Fetal Bovine Serum (FBS) (Gibco®, catalog number: 10270106 )
- Lipopolysaccharide from Escherichia coli 026:B6 (10 mg) (Sigma-Aldrich, catalog number: L2654 )
- Lucifer Yellow CH dilithium salt (25 mg) (Sigma-Aldrich, catalog number: L0259 )
- DQTM Ovalbumin (1 mg) (Molecular Probes®, catalog number: D-12053 )
- Ficoll-Paque PLUS (General Electric Company, catalog number: 17-1440-03 )
- GMP Recombinant Human Interleukin-4 (50 μg, 13 x 106 IU/mg) (Gentaur Molecular Products, catalog number: 04-GMPhuIL4-50 μg )
- Recombinant Human GM-CSF (300 μg, 3.88 x 106 IU/vial) (Gentaur Molecular Products, catalog number: 04- RHUGM-CSF-300 μg )
- IL-4
- Bovine Serum Albumin Fraction V (BSA) (Roche Diagnostics, catalog number: 10735078001 )
- FITC-conjugated anti-CD14 (RMO52) (Beckman Coulter, catalog number: IM0645U )
- FITC-conjugated anti-IgG2a (7T4-1F5) (Beckman Coulter, catalog number: IM0645U)
- Perfect-Count MicrospheresTM (Cytognos S. L., catalog number: CYT-PCM-50 )
- NaN3 (Sigma-Aldrich, catalog number: 71289 )
- FACS buffer (see Recipes)
- Complete medium (see Recipes)
- DQ-OVA (1 mg/ml) (see Recipes)
- Lucifer Yellow (10 mg/ml) (see Recipes)
- rHuIL-4 (500 IU/ml) (see Recipes)
- rHuGM-CSF (800 IU/ml) (see Recipes)
- LPS (1 mg/ml) (see Recipes)
Equipment
- 15 ml Ficoll-Paque PLUS
- 60-mm cell culture plates (Corning, catalog number: 15 430166 )
- Cytometer tubes (BD Falcon tubes, round-bottom) (Becton, Dickinson and Company, catalog number: 352052 )
- Centrifuge Heraeus Multifuge 3 L-R (Heraeus Holding, catalog number: 75004370 )
- 37 °C, 5% CO2 cell culture incubator
- BD FACSCalibur flow cytometer (Becton, Dickinson and Company, catalog number: 342975 )
Software
- CellQuest Pro software (Becton, Dickinson and Company, catalog number: 643436 )
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Luque, A., Cárdenas-Brito, S., Olivar, R. and Aran, J. M. (2013). Assessment of Human Dendritic Cell Antigen Uptake by Flow Cytometry. Bio-protocol 3(22): e974. DOI: 10.21769/BioProtoc.974.
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分类
免疫学 > 免疫细胞功能 > 树突细胞
细胞生物学 > 基于细胞的分析方法 > 流式细胞术
细胞生物学 > 基于细胞的分析方法 > 细胞吞排作用
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