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An Assay to Determine Phagocytosis of Apoptotic Cells by Cardiac Macrophages and Cardiac Myofibroblasts

一种测定心肌巨噬细胞和心肌成纤维细胞对凋亡细胞吞噬作用的方法

YH Yuma Horii
SM Shoichi Matsuda
KW Kenji Watari
AN Akiomi Nagasaka
HK Hitoshi Kurose
MN Michio Nakaya

发布: 2017年09月20日第7卷第18期 DOI: 10.21769/BioProtoc.2553 浏览次数: 9669

评审: Jia LiShahzada KhanAnonymous reviewer(s)

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Abstract

In myocardial infarction (MI), a number of cardiomyocytes undergo apoptosis. These apoptotic cardiomyocytes are promptly engulfed by phagocytes. If the dead cells are not engulfed, their noxious contents are released outside, resulting in induction of inflammation. Therefore, the removal of these dead cells is necessary. However, the contribution of each phagocyte type to the removal of apoptotic cells in infarcted hearts remains unresolved. Here, we describe an in vitro protocol for a phagocytosis assay to compare the engulfment ability of cardiac macrophages and cardiac myofibroblasts.

Keywords: Phagocytosis assay (吞噬分析) search Myofibroblast (肌成纤维细胞) search Engulfment (吞噬) search Apoptosis (细胞凋亡) search Myocardial infarction (心肌梗死) search

Background

It has long been believed that the apoptotic cells generated in failed hearts are eliminated by cardiac macrophages. However, we found that cardiac myofibroblasts, which are responsible for tissue fibrosis, also have the ability to engulf apoptotic cells after MI (Nakaya et al., 2017). The discovery prompted us to compare the engulfment ability of cardiac macrophages and cardiac myofibroblasts. Herein, we provide a detailed protocol for an in vitro phagocytosis assay to evaluate the extent of phagocytic engulfment.

Materials and Reagents

  1. Pipette tips, 1,000 µl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2179-HR )
  2. Pipette tips, 200 µl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2069-HR )
  3. Pipette tips, 20 µl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2149P-HR )
  4. Pipette tips, 10 µl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2140-HR )
  5. Surgical tape (3M, catalog number: 1527-0 )
  6. 8-0 braided silk (NATSUME SEISAKUSHO, catalog number: M6-80B2 )
  7. 5-0 braided silk (NATSUME SEISAKUSHO, catalog number: ER12-50B1 )
  8. 10 ml syringe (TERUMO, catalog number: SS-10ESZ )
  9. 23-gauge needle (TERUMO, catalog number: NN-2332R )
  10. 50 ml tube (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339652 )
  11. 6 cm dish (Corning, catalog number: 430589 )
  12. Surgical lancet (Akiyama Medical MFG, catalog number: FB10 )
  13. 70 µm EASYstrainerTM (Greiner Bio One International, catalog number: 542070 )
  14. 10 cm non-treated dish (Corning, catalog number: 430591 )
  15. 15 ml tube (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339650 )
  16. 8-well slide chamber (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 154534 )
  17. Frosted glass slides (Matsunami Glass, catalog number: S2112 )
  18. Cover glass (Matsunami Glass, catalog number: C024601 )
  19. 40 µm EASYstrainerTM (Greiner Bio One International, catalog number: 542040 )
  20. Aluminum foil
  21. 1.5 ml tube (BMBio, catalog number: BM-15 )
  22. 0.22 µm Minisart® filter (Sartorius, catalog number: 16534-K )
  23. Wild type C57BL/6JJmsSlc mouse (Japan SLC)
  24. Pentobarbital (Somnopentyl) (Kyoritsu Seiyaku, catalog number: SOM02-YA1312 )
  25. Water (NACALAI TESQUE, catalog number: 06442-95 )
  26. Phosphate buffered saline (PBS) (NACALAI TESQUE, catalog number: 14249-95 )
  27. Red blood cell (RBC) lysis buffer (Roche Diagnostics, catalog number: 11814389001 )
  28. Trypsin/Ethylenediaminetetraacetic acid (EDTA) (NACALAI TESQUE, catalog number: 35554-64 )
  29. Fixable Viability Dye eFluorTM 780 (Thermo Fisher Scientific, eBioscienceTM, catalog number: 65-0865-14 )
  30. Sevoflurane (Wako Pure Chemical Industries, catalog number: 193-17791 )
  31. Paraformaldehyde (PFA) (NACALAI TESQUE, catalog number: 26126-25 )
  32. 4’,6-Diamidino-2-phenylindole (DAPI) (Dojindo, catalog number: 340-07971 )
  33. FluorSaveTM (Merck, catalog number: 345789 )
  34. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153-100G )
  35. Trypsin (Sigma-Aldrich, catalog number: T4799-5G )
  36. Collagenase A (Roche Diagnostics, catalog number: 10103586001 )
  37. Serum-free DMEM (NACALAI TESQUE, catalog number: 08458-16 )
  38. Fatal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437028 )
  39. Penicillin-streptomycin (NACALAI TESQUE, catalog number: 09367-34 )
  40. Dispase (Roche Diagnostics, catalog number: 04942078001 )
  41. EDTA·2Na (Dojindo, catalog number: 345-01865 )
  42. FITC-conjugated anti-Ly6C antibody (BioLegend, catalog number: 128006 )
  43. PE-conjugated anti-Ly6G antibody (BioLegend, catalog number: 127607 )
  44. APC-conjugated anti-F4/80 antibody (BioLegend, catalog number: 123116 )
  45. PerCP/Cy5.5-conjugated anti-CD11b antibody (BioLegend, catalog number: 101230 )
  46. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
  47. CellTrackerTM Green 5-chloromethylfluorescein diacetate (CMFDA) dye (Thermo Fisher Scientific, InvitrogenTM, catalog number: C7025 )
  48. Poly-L-lysine solution (Sigma-Aldrich, catalog number: P4707 )
  49. Dexamethasone (Sigma-Aldrich, catalog number: D1756-25MG )
  50. Collagenase A solution (see Recipes)
  51. Culture medium (see Recipes)
  52. Dispase solution (see Recipes)
  53. FACS buffer (see Recipes)
  54. Primary antibodies (see Recipes)
  55. 8-well slide chamber coated with poly-L-lysine (see Recipes)
  56. 10 mM CMFDA dye (see Recipes)
  57. 10 mM dexamethasone (see Recipes)

Equipment

  1. Pipettes 1,000 µl (Gilson, catalog number: F123602 )
  2. Pipettes 200 µl (Gilson, catalog number: F123601 )
  3. Pipettes 20 µl (Gilson, catalog number: F123600 )
  4. Pipettes 2 µl (Gilson, catalog number: F144801 )
  5. Respirator (Shinano Manufacturing, catalog number: SN-480-7X2T )
  6. Optical microscope (Olympus, model: SZX7 )
  7. Surgical tools such as tweezers and small scissors (tools can be purchased from NATSUME SEISAKUSHO and MEISTER)
  8. Clean bench (Panasonic Healthcare, model: MCV-B131S )
  9. Water bath (TAITEC, model: Personal-11 )
  10. Centrifuge (TOMY SEIKO, model: LC-200 )
  11. CO2 incubator (SANYO, model: MCO-18AIC )
  12. Cell sorter (BD, BD Bioscience, model: FACSARIA III )
  13. Fluorescence microscope (KEYENCE, model: BZ-9000 )

Software

  1. BZ-II image analysis application (KEYENCE CORPORATION)

Procedure

English
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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). An Assay to Determine Phagocytosis of Apoptotic Cells by Cardiac Macrophages and Cardiac Myofibroblasts. Bio-protocol 7(18): e2553. DOI: 10.21769/BioProtoc.2553.

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分类

免疫学 > 免疫细胞分离 > 巨噬细胞

免疫学 > 免疫细胞功能 > 巨噬细胞

细胞生物学 > 细胞分离和培养 > 共培养

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