抗原递呈细胞的分离和体内转移

Pooja  Arora; Shalu  Sharma  Kharkwal; Steven  A.  Porcelli

doi:10.21769/BioProtoc.1260

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

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 email

发布: 2014年10月05日第4卷第19期 DOI: 10.21769/BioProtoc.1260 浏览次数: 12713

评审: Jia LiSavita NairAnonymous reviewer(s)

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Immunity, featuring study using the protocol.

Jan 2014

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

使用康可藻红素刺激冷冻保存的猪外周单个核细胞进行增殖检测，并结合FCS ExpressTM 7.18软件分析

Marlene Bravo-Parra [...] Luis G. Giménez-Lirola

2025年06月05日 1253 阅读

利用小鼠脾细胞分离并离体检测CD8⁺ T细胞的分裂与激活

Melissa Dolan [...] John M.L. Ebos

2025年08月20日 1287 阅读

研究免疫调控血管功能的新实验方法：小鼠主动脉与T淋巴细胞或巨噬细胞的共培养

Taylor C. Kress [...] Eric J. Belin de Chantemèle

2025年09月05日 952 阅读

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, CD8α^Pos dendritc cells (DCs) and CD8α^Neg 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 CD8α^Pos and CD8α^Neg 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)

Antigen presenting cell (APC) (抗原提呈细胞（APC）)

Dendritic Cell (树突状细胞)

Purification (净化)

Antigen pulsing (Antigen脉冲)

Materials and Reagents

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

Equipment

1 ml syringes (BD, catalog number: 26048 )
23 G1 needle (BD, catalog number: 305145 )
100 mm Petri dishes (Thermo Fisher Scientific, catalog number: 0875712 )
Surgical instruments (Kent Scientific, catalog number: INSMOUSEKIT )
Cell strainer (70 µm) (BD, catalog number: 352350 )
Large Petri plates (Thermo Fisher Scientific, catalog number: FB0875712 )
Vacuum filtration system (500 ml, 0.22 um) (Corning, catalog number: 431097 )
LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
Magnetic stand MACS separator (Miltenyi Biotec, catalog number: 130-042-302 )
Wide-bore 200 μl pipette tips (PerkinElmer, catalog number: 111623 )
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

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.