肿瘤相关巨噬细胞（TAM）和肿瘤相关树突细胞（TADC）的纯化

Damya Laoui; Eva Van Overmeire; Jiri Keirsse; Kiavash Movahedi; Jo A Van Ginderachter

doi:10.21769/BioProtoc.1294

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Peer-reviewed

Purification of Tumor-Associated Macrophages (TAM) and Tumor-Associated Dendritic Cells (TADC)

肿瘤相关巨噬细胞（TAM）和肿瘤相关树突细胞（TADC）的纯化

JG Jo A Van Ginderachter email

发布: 2014年11月20日第4卷第22期 DOI: 10.21769/BioProtoc.1294 浏览次数: 27615

评审: Hsin-Yi ChangHongLok LungAnonymous reviewer(s)

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Cover of Cancer Research, featuring study using the protocol.

Jan 2014

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Abstract

Tumors are heterogeneous microenvironments where complex interactions take place between neoplastic cells and infiltrating inflammatory cells, such as tumor-associated macrophages (TAM) and tumor-associated dendritic cells (TADC). The relevance of tumor-infiltrating mononuclear myeloid cells is underscored by clinical studies showing a correlation between their abundance and poor prognosis (Laoui et al., 2011). These cells are able to promote tumor progression via several mechanisms, including induction of angiogenesis, remodeling of the extracellular matrix, stimulation of cancer cell proliferation and metastasis and the inhibition of adaptive immunity (Laoui et al., 2011). Moreover, mononuclear myeloid cells are characterized by plasticity and versatility in response to microenvironmental signals, resulting in different activation states, as illustrated by the presence of distinct functional TAM subsets in tumors (Movahedi et al., 2010; Laoui et al., 2014). Here, we describe a valuable isolation technique for TAM and TADC permitting their molecular and functional characterization.

Keywords: Tumor-associated macrophages (肿瘤相关巨噬细胞)

Tumor-associated dendritic cells (树突细胞肿瘤相关)

Tumor single cell suspension (肿瘤单细胞悬液)

Macrophage purification (巨噬细胞的分离纯化)

Dendritic cell purification (树突细胞的纯化)

Materials and Reagents

Mouse
RPMI-1640 medium (RPMI) (Life Technologies, catalog number: 52400-041 )
Collagenase I (Worthington Biochemical, catalog number: LS004214 )
Collagenase IV (Worthington Biochemical, catalog number: LS004209 )
DNase I (Worthington Biochemical, catalog number: LS002060 )
Hank’s buffered salt solution (HBSS) (Life Technologies, Gibco^®)
NH₄Cl (Merck KGaA)
KHCO₃ (Merck KGaA)
EDTA (Duchefa Biochemie)
Fetal calf serum (FCS) (Life Technologies, Gibco^®)
Lymphoprep (Axis-shield, catalog number: 1114547 )
LS colums (Miltenyi Biotec, catalog number: 130-042-401 )
Anti-CD11b microbeads (Miltenyi Biotec, catalog number: 130-049-601 )
Anti-CD11c microbeads (Miltenyi Biotec, catalog number: 130-052-001 )
Purified CD16/CD32 (FcBlock) (clone 2.4G2) (BD Biosciences, catalog number: 553142 )
PE-Cy7-conjugated anti-CD11b antibody (clone M1/70) (BD Biosciences, catalog number: 552850 )
AF647-conjugated anti-Ly6C antibody (clone ER-MP20) (Bio-Rad Laboratories, AbD Serotec^®, catalog number: MCA2389A647 )
PerCP-Cy5.5-conjugated anti-I-A/I-E (MHC-II) antibody (clone M5/114.15.2) (BioLegend, catalog number: 107626 )
FITC-conjugated anti-Ly6G antibody (clone 1A8) (BD Biosciences, catalog number: 551460 )
PE-conjugated anti-SiglecF antibody (clone E50-2440) (BD Biosciences, catalog number: 552126 )
PE-conjugated anti-CD11c antibody (clone HL3) (BD Biosciences, catalog number: 553802 )
Tumor digestion medium (see Recipes)
Erythrocyte lysis buffer (see Recipes)
MACS buffer (see Recipes)
Sorting buffer (see Recipes)
Complete medium (see Recipes)

Equipment

Polyester filters cut in 10 x 10 cm squares (thread diameter 70 μm) (Spectrum^® Laboratories, catalog number: 146490 )
6-well plates (Greiner Bio-One GmbH, catalog number: 657185 )
10 ml syringes (Omnifix, catalog number: 473203 )
BD Falcon 50 ml polypropylene tubes (BD Biosciences, catalog number: 2070 )
BD Falcon 15 ml polypropylene tubes (BD Biosciences, catalog number: 2096 )
BD Falcon 5 ml polypropylene round-bottom tube (BD Biosciences, catalog number: 352063 )
Sterile culture hood
Surgical scissors and forceps
37 °C, 5% CO₂ cell culture incubator
Pipettes
Centrifuges
Shaker
Microscope
MidiMACS^TM Separator and MultiStand (Miltenyi Biotec, catalog number: 130-042-301 )
Multicolour FACS sorter (BD Biosciences, Aria flow cytometer)

Procedure

English

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如何引用

Laoui, D., Overmeire, E. V., Keirsse, J., Movahedi, K. and Ginderachter, J. A. V. (2014). Purification of Tumor-Associated Macrophages (TAM) and Tumor-Associated Dendritic Cells (TADC). Bio-protocol 4(22): e1294. DOI: 10.21769/BioProtoc.1294.