Phagocytosis Assay of Necroptotic Cells by Cardiac Myofibroblasts
心肌成纤维细胞对坏死凋亡细胞的吞噬作用分析
发布: 2017年09月20日第7卷第18期 DOI: 10.21769/BioProtoc.2552 浏览次数: 7471
评审: Jia LiSuprabhat MukherjeeAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jan 2017
Abstract
In myocardial infarction (MI), a plenty of cardiomyocytes undergo necrosis and necroptosis due to the lack of oxygen and nutrients. The dead cardiomyocytes are promptly engulfed by phagocytes. When the dead cells are not engulfed, the noxious contents of the cells are released outside, and thus, induce inflammation, and obstruct the function of organs. Therefore, phagocytosis is crucial for maintaining homeostasis of organs. Herein, we describe a protocol of an in vitro phagocytosis assay of necroptotic cells.
Keywords: Phagocytosis assay (吞噬分析)
Myofibroblast (肌成纤维细胞)
Engulfment (吞噬)
Necrosis (坏死)
Myocardial infarction (心肌梗死)
Isolation (分离)
Background
Previously, necrotic and necroptotic cells were believed to be eliminated only by cardiac macrophages in failed hearts. However, we found that cardiac myofibroblasts, which are responsible for tissue fibrosis, engulf dead cells after an MI (Nakaya et al., 2017). Herein, we provide a detailed protocol for an in vitro phagocytosis assay of necroptotic cells, employing L929 cells that undergo necroptosis by TNF-α stimulation in a caspase-3 inhibitor, Z-VAD-FMK.
Materials and Reagents
- Pipette tips, 1,000 µl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2179-HR )
- Pipette tips, 200 µl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2069-HR )
- Pipette tips, 20 µl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2149P-HR )
- Pipette tips, 10 µl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2140-HR )
- Surgical tape (3M, catalog number: 1527-0 )
- 8-0 braided silk (NATSUME SEISAKUSHO, catalog number: M6-80B2 )
- 5-0 braided silk (NATSUME SEISAKUSHO, catalog number: ER12-50B1 )
- 10 ml syringe (TERUMO, catalog number: SS-10ESZ )
- 23-gauge needle (TERUMO, catalog number: NN-2332R )
- 50 ml tube (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339652 )
- 6 cm dish (Corning, catalog number: 430589 )
- Surgical lancet (Akiyama Medical MFG, catalog number: FB10 )
- 70 µm EASYstrainerTM (Greiner Bio One International, catalog number: 542070 )
- 10-cm non-treated dish (Corning, catalog number: 430591 )
- 15 ml tube (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339650 )
- 8-well slide chamber (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 154534 )
- Cover glass (Matsunami Glass, catalog number: C024601 )
- 0.22 µm Minisart® filter (Sartorius, catalog number: 16534-K )
- Wild type C57BL/6JJmsSlc mouse (Japan SLC)
- L929 cells (National Institutes of Biomedical Innovation, Health and Nutrition, Japanese Collection of Research Bioresources Cell Bank, catalog number: JCRB9003 )
- Pentobarbital (Somnopentyl) (Kyoritsu Seiyaku, catalog number: SOM02-YA1312 )
- Phosphate buffered saline (PBS) (NACALAI TESQUE, catalog number: 14249-95 )
- Red blood cell (RBC) lysis buffer (Roche Diagnostics, catalog number: 11814389001 )
- Trypsin/Ethylenediaminetetraacetic acid (EDTA) (NACALAI TESQUE, catalog number: 35554-64 )
- Paraformaldehyde (PFA) (NACALAI TESQUE, catalog number: 26126-25 )
- 4’,6-Diamidino-2-phenylindole (DAPI) (Dojindo, catalog number: 340-07971 )
- FluorSaveTM (Merck, catalog number: 345789 )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153-100G )
- Trypsin (Sigma-Aldrich, catalog number: T4799-5G )
- Collagenase A (Roche Diagnostics, catalog number: 10103586001 )
- Serum-free DMEM (NACALAI TESQUE, catalog number: 08458-16 )
- Penicillin streptomycin (NACALAI TESQUE, catalog number: 09367-34 )
- Dimethyl sulphoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
- CellTrackerTM Green 5-chloromethylfluorescein diacetate (CMFDA) dye (Thermo Fisher Scientific, InvitrogenTM, catalog number: C7025 )
- Z-VAD-FMK (Z-Val-Aal-Asp(OMe)-CH2F) (PEPTIDE INSTITUTE, catalog number: 3188-v )
- hTNF-α (PeproTech, catalog number: 300-01A )
- Poly-L-lysine solution (Sigma-Aldrich, catalog number: P4707 )
- Water (NACALAI TESQUE, catalog number: 06442-95 )
- Fatal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437028 )
- Collagenase A solution (see Recipes)
- Culture medium (see Recipes)
- 10 mM CMFDA dye (see Recipes)
- 10 mM Z-VAD-FMK (see Recipes)
- 10 µg/ml hTNF-α solution (see Recipes)
- 8-well slide chamber coated with poly-L-lysine (see Recipes)
Equipment
- Pipettes 1,000 µl (Gilson, catalog number: F123602 )
- Pipettes 200 µl (Gilson, catalog number: F123601 )
- Pipettes 20 µl (Gilson, catalog number: F123600 )
- Pipettes 2 µl (Gilson, catalog number: F144801 )
- Respirator (Shinano Manufacturing, catalog number: SN-480-7X2T )
- Optical microscope (Olympus, model: SZX7 )
- Surgical tools such as tweezers (tools can be purchased from NATSUME SEISAKUSHO and MEISTER)
- Scissors (NATSUME SEISAKUSHO, catalog number: B-12 )
- Clean bench (Panasonic Healthcare, model: MCV-B131S )
- Water bath (TAITEC, model: Personal-11 )
- Centrifuge (TOMY SEIKO, model: LC-200 )
- CO2 incubator (SANYO, model: MCO-18AIC )
- Fluorescence microscope (KEYENCE, model: BZ-9000 )
Software
- BZ-II image analysis application (KEYENCE CORPORATION)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Horii, Y., Matsuda, S., Watari, K., Nagasaka, A., Kurose, H. and Nakaya, M. (2017). Phagocytosis Assay of Necroptotic Cells by Cardiac Myofibroblasts. Bio-protocol 7(18): e2552. DOI: 10.21769/BioProtoc.2552.
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分类
免疫学 > 免疫细胞功能 > 巨噬细胞
免疫学 > 免疫细胞分离 > 巨噬细胞
细胞生物学 > 细胞分离和培养 > 共培养
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