骨髓源性单核细胞的过继转移

Damya  Laoui; Eva Van Overmeire; Chloé Abels; Jiri Keirsse; Jo A Van Ginderachter

doi:10.21769/BioProtoc.3134

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Peer-reviewed

Adoptive Transfer of Monocytes Sorted from Bone Marrow

骨髓源性单核细胞的过继转移

DL Damya Laoui email

EO Eva Van Overmeire

CA Chloé Abels

JK Jiri Keirsse

JG Jo A Van Ginderachter

发布: 2019年01月05日第9卷第1期 DOI: 10.21769/BioProtoc.3134 浏览次数: 7625

评审: Alka MehraAmriti Rajender LullaJia Li

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The authors used this protocol in:

Cover of Cancer Research, featuring study using the protocol.

Jan 2016

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Abstract

Inflammatory Ly6C^hi monocytes can give rise to distinct mononuclear myeloid cells in the tumor microenvironment, such as monocytic myeloid-derived suppressor cells (Mo-MDSC), immature macrophages, M2-like tumor-associated macrophages (TAMs), M1-like TAMs or monocyte-derived dendritic cells (Mo-DCs). This protocol describes a method to assess the fate and recruitment of inflammatory Ly6C^hi monocytes in the tumor microenvironment.

Keywords: Adoptive transfer (过继转移)

Monocytes (单核细胞)

Pre-cDCs (Pre-cDCs)

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

Tumor-associated dendritic cells (肿瘤相关树突状细胞)

Cell sorting (细胞分选)

Background

Tumors are heterogeneous microenvironments where complex interactions take place between neoplastic cells and infiltrating inflammatory cells, such as tumor-associated macrophages (TAMs) and tumor-associated dendritic cells (TADCs). The relevance of tumor-infiltrating mononuclear myeloid cells is underscored by clinical studies showing a correlation between their abundance and poor prognosis (Bolli et al., 2007). The origin of TAMs and TADCs has been a matter of debate, since several levels of complexity result in considerable TAM and TADC heterogeneity (Movahedi et al., 2010; Laoui et al., 2014, Laoui et al., 2016; Van Overmeire et al., 2016; Kiss et al., 2018). Here, we describe a valuable method to adoptively transfer bone-marrow derived monocytes permitting the assessment of their recruitment and fate in tumors.

Materials and Reagents

Polyester filters cut in 10 x 10 cm squares, thread diameter 70 μm (Specturmlabs, catalog number: 146490)
10 ml syringes (Omnifix, catalog number: 473203)
1 ml syringes (Greiner, catalog number: 470203)
27 G needles (BD Bioscience, catalog number: 300635)
25 G needles (BD Biosciences, catalog number: 300400)
19 G needles (BD Biosciences, catalog number: 301500)
Falcon standard tissue culture dish (Fisher Scientific, catalog number: 353003)
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)
70 µm sterile nylon gauze
LS columns (Miltenyi, catalog number: 130-042-401)
Naive mice: Age preferably between 6 and 12 weeks, strain can vary depending on the experiment/project (in this example we used C57BL/6 mice)
Ethanol absolute analaR Normapur ACS (VWR Chemicals, catalog number: 84857360)
RPMI-1640 medium (RPMI) (Life Technologies, catalog number: 52400-041)
Fetal calf serum (FCS) (Life Technologies, Gibco, catalog number: DE14-801F)
L-glutamine (Life Technologies, catalog number: 25030-024)
Penicillin-streptomycin (Life Technologies, catalog number: 15140-130)
Ammonium chloride (NH₄Cl) (Merck KGaA, catalog number: 1011450500)
Potassium bicarbonate (KHCO₃) (Merck KGaA, catalog number: 104852)
EDTA (Duchefa Biochemie, catalog number: E0511.1000)
Hank’s buffered salt solution (HBSS) (Life Technologies, Gibco, catalog number: 14175129)
Anti-CD11b microbeads (Miltenyi, catalog number: 130-049-601)
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) (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)
APC-Cy7-conjugated anti CD45 (clone 30-F11) (BioLegend, catalog number: 103116)
CellTrace Violet (Thermo Fisher Scientific, Molecular probes^TM, catalog number: C34557)
Trypan blue (Life Technologies, Gibco, catalog number: 15250061)
DMSO
70% ethanol (see Recipes)
Complete medium (see Recipes)
Erythrocyte lysis buffer (see Recipes)
MACS buffer (see Recipes)
Sorting buffer (see Recipes)
Violet tracer (see Recipes)

Equipment

Sterile culture hood, PSM Optimale 18 (ADS)
Surgical scissors and forceps
37 °C, 5% CO₂ cell culture incubator (Binder, VWR)
Pipettes (Gilson)
Centrifuges 5810 R (Eppendorf, model: 5810 R)
Shaker KS 260 Basic (IKA, model: KS 260 basic)
Microscope Eclipse TS100 (Nikon, model: Eclipse TS100)
MidiMACS^TM Separator and MultiStand (Miltenyi, catalog number: 130-042-301)
Multicolor FACS Sorter-FACS Aria II (BD Biosciences Aria flow cytometer)

Procedure

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如何引用

Laoui, D., Overmeire, E. V., Abels, C., Keirsse, J. and Ginderachter, J. A. V. (2019). Adoptive Transfer of Monocytes Sorted from Bone Marrow. Bio-protocol 9(1): e3134. DOI: 10.21769/BioProtoc.3134.