提取和利用凋亡Jurkat细胞诱导腹腔巨噬细胞分泌白介素-10

Mei Song; Xiaojing   Ma

doi:10.21769/BioProtoc.3467

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Isolation and Stimulation of Peritoneal Macrophages with Apoptotic Jurkat Cells to Produce IL-10

提取和利用凋亡Jurkat细胞诱导腹腔巨噬细胞分泌白介素-10

MS Mei Song

Xiaojing Ma email

发布: 2019年12月20日第9卷第24期 DOI: 10.21769/BioProtoc.3467 浏览次数: 5423

评审: Anonymous reviewer(s)

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The authors used this protocol in:

Cover of Immunity, featuring study using the protocol.

Dec 2007

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Abstract

Clearance of apoptotic cells by macrophages is critical to ensuring cellular homeostasis and suppression of autoimmunity. Macrophage recognition of apoptotic cells triggers an anti-inflammatory response, which is mediated by the release of IL-10, TGF-β etc. with concurrent inhibition of pro-inflammatory cytokines (such as TNFα, IL-12, IL-1β). To characterize cytokine profile produced by macrophages during phagocytosis of apoptotic cells, we developed an effective, more physiologic system using isolated murine peritoneal macrophages and T-lymphocyte cell line Jurkat as a source of apoptotic cells. Apoptosis of Jurkat cells is induced with staurosporine, a protein kinase C (PKC) inhibitor and detected by Annexin V/propidium iodide staining. This in vitro assay demonstrates that murine peritoneal macrophages produce large amounts of IL-10 following exposure to apoptotic Jurkat cells.

Keywords: Apoptosis (细胞凋亡)

Background

Production of IL-10, a major immunoregulatory cytokine, by phagocytes during clearance of apoptotic cells is critical to ensuring cellular homeostasis and suppression of autoimmunity (Chung et al., 2007). Little is known about the regulatory mechanisms in this fundamental process. To elucidate the molecular mechanisms involved in the regulation of IL-10 gene expression in macrophages upon interaction with apoptotic cells, we developed this protocol to explore key regulators of IL-10 production induced by apoptotic cells.

Materials and Reagents

24-well plate (Corning, catalog number: 3563847)
T-75 flask (Corning, catalog number: 431464)
Falcon^® 50 ml High Clarity PP Centrifuge Tube (Corning, catalog number: 352070)
Posi-Click 1.7 ml microcentrifuge tube (Denville, catalog number: C2170)
10-ml syringe (BD, catalog number: 309695)
25 G and 18 G needles (BD)
C57BL/6 mice (6-8 weeks old)
Cell line: Jurkat, Clone E6-1 (ATCC, catalog number: TIB-152)
Fetal Bovine Serum (Thermo Fisher, catalog number: 16000)
RPMI 1640 Medium, GlutaMAX^TM Supplement, and HEPES (Thermo Fisher, catalog number: 72400)
Penicillin-Streptomycin (10,000 U/ml) (Thermo Fisher, catalog number: 15140)
GlutaMAX^TM Supplement (Thermo Fisher, catalog number: 35050)
Staurosporine (Sigma-Aldrich, catalog number: 19-123)
DPBS, no calcium, no magnesium (Thermo Fisher, catalog number: 14190)
FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, catalog number: 556547)
Mouse IL-10 ELISA Set (BD, catalog number: 565252)
BD BBL Dehydrated Culture Media (Fisher Scientific, catalog number: 211716)
Dimethyl sulfoxide (Sigma-Aldrich, catalog number: D2560)
70% Ethanol
Complete media for Jurkat T cells (see Recipe 1)
3% (w/v) Brewer thioglycollate medium (see Recipe 2)
1x Binding buffer(see Recipe 3)
RPMI-1640-10 (see Recipe 4)
0.5 mg/ml Staurosporine (1,000x stock) (see Recipe 5)

Equipment

Tissue culture hood (biosafety cabinet, Forma Scientific, model: 1284)
37 °C, 5% CO₂ humidified incubator (Thermo Forma, model: 3110 Series II)
Centrifuge (Thermo Scientific, ST8 Benchtop Centrifuge)
Optical microscopy (Olympus, CK30)
Flow cytometer (BD, FACSCalibur)
Sterile Surgical Scissors (Miltex, MH5-302)
Hemacytometer (Hausser Scientific, catalog number: 02-671-5)
Autoclave

Software

FlowJo software (7.6.1)
GraphPad Prism

Procedure

English

中文翻译

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如何引用

Song, M. and Ma, X. (2019). Isolation and Stimulation of Peritoneal Macrophages with Apoptotic Jurkat Cells to Produce IL-10. Bio-protocol 9(24): e3467. DOI: 10.21769/BioProtoc.3467.