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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   

编审
Jia Li
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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

  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. Cover glass (Matsunami Glass, catalog number: C024601 )
  18. 0.22 µm Minisart® filter (Sartorius, catalog number: 16534-K )
  19. Wild type C57BL/6JJmsSlc mouse (Japan SLC)
  20. L929 cells (National Institutes of Biomedical Innovation, Health and Nutrition, Japanese Collection of Research Bioresources Cell Bank, catalog number: JCRB9003 )
  21. Pentobarbital (Somnopentyl) (Kyoritsu Seiyaku, catalog number: SOM02-YA1312 )
  22. Phosphate buffered saline (PBS) (NACALAI TESQUE, catalog number: 14249-95 )
  23. Red blood cell (RBC) lysis buffer (Roche Diagnostics, catalog number: 11814389001 )
  24. Trypsin/Ethylenediaminetetraacetic acid (EDTA) (NACALAI TESQUE, catalog number: 35554-64 )
  25. Paraformaldehyde (PFA) (NACALAI TESQUE, catalog number: 26126-25 )
  26. 4’,6-Diamidino-2-phenylindole (DAPI) (Dojindo, catalog number: 340-07971 )
  27. FluorSaveTM (Merck, catalog number: 345789 )
  28. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153-100G )
  29. Trypsin (Sigma-Aldrich, catalog number: T4799-5G )
  30. Collagenase A (Roche Diagnostics, catalog number: 10103586001 )
  31. Serum-free DMEM (NACALAI TESQUE, catalog number: 08458-16 )
  32. Penicillin streptomycin (NACALAI TESQUE, catalog number: 09367-34 )
  33. Dimethyl sulphoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
  34. CellTrackerTM Green 5-chloromethylfluorescein diacetate (CMFDA) dye (Thermo Fisher Scientific, InvitrogenTM, catalog number: C7025 )
  35. Z-VAD-FMK (Z-Val-Aal-Asp(OMe)-CH2F) (PEPTIDE INSTITUTE, catalog number: 3188-v )
  36. hTNF-α (PeproTech, catalog number: 300-01A )
  37. Poly-L-lysine solution (Sigma-Aldrich, catalog number: P4707 )
  38. Water (NACALAI TESQUE, catalog number: 06442-95 )
  39. Fatal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437028 )
  40. Collagenase A solution (see Recipes)
  41. Culture medium (see Recipes)
  42. 10 mM CMFDA dye (see Recipes)
  43. 10 mM Z-VAD-FMK (see Recipes)
  44. 10 µg/ml hTNF-α solution (see Recipes)
  45. 8-well slide chamber coated with poly-L-lysine (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 (tools can be purchased from NATSUME SEISAKUSHO and MEISTER)
  8. Scissors (NATSUME SEISAKUSHO, catalog number: B-12 )
  9. Clean bench (Panasonic Healthcare, model: MCV-B131S )
  10. Water bath (TAITEC, model: Personal-11 )
  11. Centrifuge (TOMY SEIKO, model: LC-200 )
  12. CO2 incubator (SANYO, model: MCO-18AIC )
  13. Fluorescence microscope (KEYENCE, model: BZ-9000 )

Software

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

Procedure

Procedures A and B are also applicable to the assay to determine phagocytosis of apoptotic cells by cardiac macrophages and Cardiac Myofibroblasts (Horii et al., 2017).

  1. Establishment of an MI mouse model by surgical operation
    1. Prepare an 8- to 10-week-old male wild type mouse of the strain, C57BL/6J.
    2. In order to anesthetize the mouse, administer pentobarbital (50 mg/kg) via an intraperitoneal injection.
    3. Fixate the mouse on its back with surgical tape.
    4. Perform artificial respiration by using a respirator (volume of air for respiration: 0.5 cc, respiratory frequency: 120 bpm).
    5. Under an optical microscope, surgically open the chest and expose the heart using sterilized surgical tools.
    6. Perform the permanent occlusion of the left coronary artery by using 8-0 braided silk (see Video 1).
    7. Close the chest by using 5-0 braided silk.
    8. After the operation, give the mouse the appropriate treatment, and keep it warm until recovery.

      Video 1. How to establish an MI mouse model

  2. Isolation of cardiac myofibroblasts from the MI mouse model
    1. Euthanize two mice after 3 days of the MI operation by administering pentobarbital (150 mg/kg) via an intraperitoneal injection.
    2. Open the chest by using sterilized scissors to expose the heart.
    3. Cut the right atrium and prick the left ventricle with a 10 ml syringe containing 10 ml of ice-cold PBS and mounted with a 23-gauge needle.
    4. Perfuse the heart with 10 ml of ice-cold PBS.
    5. Collect the heart, and remove the atria.
    6. Put the heart in a 50 ml tube containing 10 ml of ice-cold PBS for storage, while other mice are being sacrificed.
    7. Discard the supernatant and add 5 ml of ice-cold PBS.
    8. Transfer the heart along with PBS into a 6 cm dish from the 50 ml tube on a clean bench.
    9. Cut each heart into 15 small pieces using two surgical lancets, while keeping them on ice.
    10. Discard PBS.
    11. To wash the heart pieces, sprinkle 5 ml of PBS on them.
    12. Repeat steps B10-B11.
    13. Transfer the heart pieces into a 50 ml tube, and remove PBS.
    14. Add 4 ml of collagenase A solution (see Recipes) to the tube.
    15. Incubate the tube containing the mixture of the minced hearts and collagenase A solution in a water bath at 37 °C, while shaking at 120 rpm for 10 min.
    16. Discard supernatant which contains numerous hematopoietic cells.
    17. Repeat steps B14-B16 once again.
    18. Add 4 ml of collagenase A solution to the tube which now contains the residual material.
    19. Incubate the tube containing the mixture of the minced hearts and collagenase A solution in a water bath at 120 rpm and 37 °C for 10 min.
    20. Pass the supernatant, which contains isolated cells, through a 70 µm EASYstrainerTM into a new 50 ml tube.
    21. Centrifuge the collected cell suspension at 300 x g for 5 min at room temperature, and discard supernatant.
    22. Add 1 ml of the culture medium (see Recipes) to the cell pellet, and keep it on ice.
    23. Repeat steps B18-B22, eight times in total.
    24. After eight agitations, combine all the cell suspensions (1 ml x 8) into a new 50 ml tube.
    25. Centrifuge this cell suspension at 300 x g for 5 min, and discard the supernatant after centrifugation.
    26. Suspend the cell pellet in 1 ml of RBC lysis buffer, and incubate for 1 min at room temperature.
    27. Add 9 ml of the culture medium to the cell suspension.
    28. Centrifuge the cell suspension at 300 x g for 5 min, and discard the supernatant after centrifugation.
    29. Suspend the cell pellet in 10 ml of the culture medium.
    30. Plate this cell suspension on a 10-cm non-treated dish, and incubate overnight in a 5% CO2 incubator at 37 °C.
    31. After overnight incubation, discard the culture medium, and add 10 ml of fresh culture medium.
    32. Culture the isolated cardiac cells for more than 6 days.
      Note: Myofibroblasts attach themselves to the plate; unattached contaminating cells, including hematopoietic cells, are removed by changing the culture medium. When the myofibroblasts reach pre-confluence, passage them.
    33. One day before conducting the in vitro phagocytosis assay, discard the culture medium from the dish, and wash the dish twice with 10 ml of PBS.
    34. Add 1 ml of trypsin/EDTA, and incubate at 37 °C for 1 min.
    35. Add 9 ml of the culture medium, and transfer the cell suspension into a 15-ml tube.
    36. Centrifuge the cell suspension at 300 x g for 5 min, and discard the supernatant after centrifugation.
    37. Add 10 ml of the culture medium, and suspend the cell pellet.
    38. Count the cell number, and adjust it to 1 x 105 cells/ml by adding the culture medium.
    39. Add 200 µl of this cell suspension to an 8-well slide chamber coated with poly-L-lysine (2 x 104 cells/well, see Recipes).
    40. Incubate in a 5% CO2 incubator at 37 °C overnight.

  3. Preparation of necroptotic L929 cells
    1. Seed 2.5 x 106 L929 cells on a 10 cm non-treated dish containing the culture medium.
    2. Incubate in a 5% CO2 incubator at 37 °C overnight.
    3. Aspirate the medium, and wash twice using 10 ml of serum-free DMEM.
    4. Add 10 ml of serum-free DMEM containing 10 µl of 10 mM CMFDA dye (see Recipes) (final concentration would be 10 µM).
    5. Incubate in a 5% CO2 incubator at 37 °C for 30 min.
    6. Aspirate the medium, and incubate on ice for 1 min.
    7. Wash 4 times using 10 ml of the culture medium.
    8. Add 10 ml of the culture medium containing 20 µl of 10 mM Z-VAD-FMK (see Recipes) (final concentration would be 20 µM).
    9. Incubate in a CO2 incubator for 2 h.
    10. Add 10 µl of 10 µg/ml hTNF-α solution (see Recipes) (final concentration is 10 ng/ml).
    11. Incubate in a 5% CO2 incubator at 37 °C for 4 h.
    12. Transfer the supernatant into a 50 ml tube.
      Note: The supernatant contains necroptotic L929 cells.
    13. Centrifuge the cell suspension at 300 x g for 5 min, and discard the supernatant after centrifugation.
    14. Add 10 ml of the culture medium.
    15. Repeat steps C13 and C14 thrice totally.
    16. Count the cell number.
    17. Adjust the cell number to 1 x 106 cells/ml with culture medium.

  4. Phagocytosis assay
    1. Prepare the cardiac myofibroblasts cultured with 200 µl of culture medium per well on the 8-well slide chamber in a 5% CO2 incubator at 37 °C (this slide was made in B40).
    2. Discard the supernatant.
    3. Add 200 µl of CMFDA-labeled necroptotic L929 cells at a concentration of 1 x 106 cells/ml (2 x 105 cells/well).
      Note: The number of engulfed necroptotic cells is 10 times the number of phagocytes per well.
    4. Incubate for 3 h at 37 °C in a 5% CO2 incubator.
    5. Discard the supernatant, and wash with 200 µl of PBS thrice.
      Note: Unengulfed necroptotic cells are attached to phagocytes, and these cells are removed by washing with PBS.
    6. To fix the cells, add 200 µl of 1% PFA/PBS solution, and incubate for 15 min at room temperature.
    7. Discard the supernatant, and wash with 200 µl of PBS.
    8. Discard the supernatant, and remove the chamber wall from the glass slide.
    9. Mount the slide with FluorSaveTM reagent containing 0.1% DAPI, and place a cover glass over the glass slide.
    10. Observe under phase contrast and fluorescence microscopes.
      Note: Images are captured at 20x magnification by BZ-9000 . Obtain phase contrast images as well as the images captured by using GFP-BP and DAPI-BP filters.

Data analysis

  1. Capture three types of images (phase contrast images as well as images obtained using GFP-BP and DAPI-BP filters) from 12-15 randomly selected fields (Figure 1).
  2. Merge the images using the BZ-II image analysis application.
  3. Count the number of the phagocytes and engulfed cells (CMFDA-positive and merged phagocytic cells).
    Note: In the phase contrast images, unengulfed cells can be observed in the form of light phase and blurry images, whereas engulfed cells can be observed in the form of dark phase images. Do not consider these light phase cells as engulfed cells.
  4. Calculate the phagocytosis index (Figure1).
    Note: Phagocytosis index is defined as the number of engulfed cells per phagocyte. First, count the number of nuclei of myofibroblasts that are stained by DAPI (pink arrowheads) and then count the number of engulfed necroptotic cells labeled with CMFDA (yellow arrowheads). After the counting, phagocytosis index is calculated as the number of engulfed necroptotic cells divided by the number of nuclei of myofibroblasts. For example, when the number of nuclei of myofibroblasts is 11 and the number of engulfed necroptotic cells is 5, phagocytosis index is calculated as 5/11 = 0.45. Following the calculation, phagocytosis index is averaged over 12-15 randomly selected fields.


    Figure 1. Cardiac myofibroblasts engulf the necroptotic cells. Cardiac myofibroblasts were isolated from the MI mouse models 3 days after operation. The necroptotic L929 cells are labeled with CMFDA (green). The pictures with arrowheads were rescaled to allow better visualization of phagocytosis. Count the number of phagocytes (pink arrowheads) and engulfed cells (yellow arrowheads), and calculate the phagocytic index. Scale bars = 50 µm.

Recipes

  1. Collagenase A solution
    1. Take 50 ml PBS to a 50 ml tube
    2. Add:
      50 mg of BSA
      50 mg of trypsin
      50 mg of collagenase A
    3. Mix well, and incubate at room temperature
    4. Filter the solution through a 0.22 µm Minisart® filter
  2. Culture medium
    1. Remove 55 ml of serum-free DMEM from 500 ml of serum-free DMEM
    2. Add 50 ml of FBS
    3. Add 5 ml of penicillin-streptomycin
  3. 10 mM CMFDA dye
    Add 10.76 µl of DMSO to 50 µg of CMFDA dye
  4. 10 mM Z-VAD-FMK
    Add 240 µl of DMSO to 1.1 mg of Z-VAD-FMK
  5. 10 µg/ml hTNF-α solution
    1. Add 10 ml of sterile distilled water to a 15-ml tube
    2. Add 10 mg of BSA
    3. Filter the solution through a 0.22 µm Minisart® filter and make 0.1% BSA solution
    4. Add 100 µl of 0.1% BSA solution to 10 µg of hTNF-α and make 100 µg/ml of hTNF-α solution
    5. Add 90 µl of 0.1% BSA solution to 10 µl of 100 µg/ml of hTNF-α solution
  6. 8-well slide chamber coated with poly-L-lysine
    1. Add 200 µl of poly-L-lysine solution to the 8-well slide chamber
    2. Incubate in a CO2 incubator for 3 h
    3. Discard the poly-L-lysine solution
    4. Wash using PBS

Acknowledgments

When using this protocol, please refer to M Nakaya et al. (2017) J Clin Invest. Funding was provided by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) [to M.N (17H03984)]; from Grant-in-Aid for Scientific Research on Innovative Areas (Homeostatic regulation by various types of cell death) from MEXT [to M.N (17H05510)]. All animal experiments were performed using approved protocols and in accordance with the guidelines of Kyushu University.

References

  1. 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 Protoc 7(18): e2553.
  2. Nakaya, M., Watari, K., Tajima, M., Nakaya, T., Matsuda, S., Ohara, H., Nishihara, H., Yamaguchi, H., Hashimoto, A., Nishida, M., Nagasaka, A., Horii, Y., Ono, H., Iribe, G., Inoue, R., Tsuda, M., Inoue, K., Tanaka, A., Kuroda, M., Nagata, S. and Kurose, H. (2017). Cardiac myofibroblast engulfment of dead cells facilitates recovery after myocardial infarction. J Clin Invest 127(1): 383-401.

简介

在心肌梗死(MI)中,由于缺氧和营养,大量心肌细胞发生坏死和坏死。 死亡心肌细胞迅速吞噬吞噬细胞。 当死细胞不被吞没时,细胞的有害物质被释放到外部,从而引起炎症,并阻碍器官的功能。 因此,吞噬对维持器官的体内平衡至关重要。 在这里,我们描述了一种神经细胞的体外吞噬作用测定方案。
【背景】以前,认为坏死性坏死细胞和坏死核细胞只能通过心脏巨噬细胞在失败的心脏中消除。 然而,我们发现负责组织纤维化的心脏肌成纤维细胞在MI后吞噬死细胞(Nakaya等,2017)。 本文中,我们提供了一种细胞凋亡细胞的体外吞噬作用的详细方案,采用在caspase-3抑制剂Z-VAD-FMK中通过TNF-α刺激进行人脑坏死的L929细胞。

关键字:吞噬分析, 肌成纤维细胞, 吞噬, 坏死, 心肌梗死, 分离

材料和试剂

  1. 移液管吸头,1,000μl(Thermo Fisher Scientific,Thermo Scientific TM,目录号:2179-HR)
  2. 移液瓶头,200μl(Thermo Fisher Scientific,Thermo Scientific TM,目录号:2069-HR)
  3. 移液瓶头,20μl(Thermo Fisher Scientific,Thermo Scientific TM,目录号:2149P-HR)
  4. 移液瓶头,10μl(Thermo Fisher Scientific,Thermo Scientific TM,目录号:2140-HR)
  5. 手术胶带(3M,目录号:1527-0)
  6. 8-0编织丝(NATSUME SEISAKUSHO,目录号:M6-80B2)
  7. 5-0编织丝(NATSUME SEISAKUSHO,目录号:ER12-50B1)
  8. 10ml注射器(TERUMO,目录号:SS-10ESZ)
  9. 23号针(TERUMO,目录号:NN-2332R)
  10. (Thermo Fisher Scientific,Thermo Scientific TM ,目录号:339652)
  11. 6厘米盘(康宁,目录号:430589)
  12. 手术刺血针(秋山医疗MFG,目录号:FB10)
  13. 70μmEASYstrainer TM (Greiner Bio One International,目录号:542070)
  14. 10厘米未经处理的菜(Corning,目录号:430591)
  15. (Thermo Fisher Scientific,Thermo Scientific TM ,目录号:339650)
  16. 8孔滑动室(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:154534)
  17. 盖玻璃(松本玻璃,目录号:C024601)
  18. 0.22μmMinisart ®过滤器(Sartorius,目录号:16534-K)
  19. 野生型C57BL / 6JJmsSlc小鼠(日本SLC)
  20. L929细胞(National Institutes of Biomedical Innovation,Health and Nutrition,Japanese Collection of Research Bioresources Cell Bank,目录号:JCRB9003)
  21. 戊巴比妥(Somnopentyl)(Kyoritsu Seiyaku,目录号:SOM02-YA1312)
  22. 磷酸盐缓冲盐水(PBS)(NACALAI TESQUE,目录号:14249-95)
  23. 红细胞(RBC)裂解缓冲液(Roche Diagnostics,目录号:11814389001)
  24. 胰蛋白酶/乙二胺四乙酸(EDTA)(NACALAI TESQUE,目录号:35554-64)
  25. 多聚甲醛(PFA)(NACALAI TESQUE,目录号:26126-25)
  26. 4',6-二脒基-2-苯基吲哚(DAPI)(Dojindo,目录号:340-07971)
  27. FluorSave TM (默克,目录号:345789)
  28. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153-100G)
  29. 胰蛋白酶(Sigma-Aldrich,目录号:T4799-5G)
  30. 胶原酶A(Roche Diagnostics,目录号:10103586001)
  31. 无血清DMEM(NACALAI TESQUE,目录号:08458-16)
  32. 青霉素链霉素(NACALAI TESQUE,目录号:09367-34)
  33. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2650)
  34. CellTracker 绿色5-氯甲基荧光素二乙酸酯(CMFDA)染料(Thermo Fisher Scientific,Invitrogen TM,目录号:C7025)
  35. Z-VAD-FMK(Z-Val-Aal-Asp(OMe)-CH 2 F)(PEPTIDE INSTITUTE,目录号:3188-v)
  36. hTNF-α(PeproTech,目录号:300-01A)
  37. 聚-L-赖氨酸溶液(Sigma-Aldrich,目录号:P4707)
  38. 水(NACALAI TESQUE,目录号:06442-95)
  39. 致命的牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10437028)
  40. 胶原酶A溶液(参见食谱)
  41. 培养基(见食谱)
  42. 10 mM CMFDA染料(见食谱)
  43. 10 mM Z-VAD-FMK(见配方)
  44. 10μg/ ml hTNF-α溶液(参见食谱)
  45. 涂有聚-L-赖氨酸的8孔滑动室(参见食谱)

设备

  1. 移液管1000μl(Gilson,目录号:F123602)
  2. 移液器200μl(Gilson,目录号:F123601)
  3. 移液器20μl(Gilson,目录号:F123600)
  4. 移液2μl(Gilson,目录号:F144801)
  5. 呼吸器(Shinano Manufacturing,目录号:SN-480-7X2T)
  6. 光学显微镜(Olympus,型号:SZX7)
  7. 手术工具如镊子(工具可以从NATSUME SEISAKUSHO和MEISTER购买)
  8. 剪刀(NATSUME SEISAKUSHO,目录号:B-12)
  9. 洁净台(Panasonic Healthcare,型号:MCV-B131S)
  10. 水浴(TAITEC,型号:Personal-11)
  11. 离心机(TOMY SEIKO,型号:LC-200)
  12. CO 2培养箱(SANYO,型号:MCO-18AIC)
  13. 荧光显微镜(KEYENCE,型号:BZ-9000)

软件

  1. BZ-II图像分析应用(KEYENCE CORPORATION)

程序

方法A和B也适用于通过心脏巨噬细胞和心肌成纤维细胞(Horii等人,2017)测定凋亡细胞吞噬作用的测定法。

  1. 通过外科手术建立MI小鼠模型
    1. 准备8至10周龄的雄性野生型小鼠C57BL / 6J。
    2. 为了麻醉小鼠,腹腔注射戊巴比妥(50mg / kg)。
    3. 用手术胶带将鼠标固定在背面。
    4. 使用呼吸器进行人工呼吸(呼吸空气量:0.5cc,呼吸频率:120 bpm)。
    5. 在光学显微镜下,手术打开胸部并用无菌手术工具暴露心脏
    6. 使用8-0编织丝来执行左冠状动脉的永久闭塞(见视频1)
    7. 使用5-0编织丝来关闭胸部。
    8. 手术后,给予老鼠适当的治疗,并保持温暖,直到康复。

      Video 1. How to establish an MI mouse model

      To play the video, you need to install a newer version of Adobe Flash Player.

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  2. 从MI小鼠模型中分离心肌肌成纤维细胞
    1. 通过腹膜内注射施用戊巴比妥(150mg / kg),在MI手术3天后使两只小鼠安乐死。
    2. 用无菌剪刀打开胸部露出心脏。
    3. 切开右心房,用10ml注射器装入10ml冰冷的PBS,并用23号针头装上左心室。
    4. 用10ml冰冷的PBS清洗心脏。
    5. 收集心脏,并去除心房。
    6. 将心放在含有10ml冰冷PBS的50ml管中,以保存其他小鼠。
    7. 弃上清液,加入5ml冰冷的PBS
    8. 将心脏与PBS一起转移到干净的工作台上的50ml管中的6厘米盘中
    9. 用两只手术刀将每颗心脏切成15个小块,同时将其保持在冰上。
    10. 丢弃PBS。
    11. 洗心粉,洒上5ml的PBS。
    12. 重复步骤B10-B11。
    13. 将心脏碎片转移到50ml管中,并移除PBS。
    14. 加入4ml胶原酶A溶液(参见食谱)到管中
    15. 将包含切碎的心脏和胶原酶A溶液的混合物的管在37℃的水浴中孵育,同时以120rpm摇动10分钟。
    16. 丢弃含有许多造血细胞的上清液
    17. 再次重复步骤B14-B16。
    18. 将4ml胶原酶A溶液加入到现在含有残留物质的管中
    19. 将含有切碎的心脏和胶原酶A溶液的混合物的管在120rpm和37℃的水浴中孵育10分钟。
    20. 将含有分离细胞的上清液通过70μmEASYstrainer TM 通入新的50ml管中。
    21. 在室温下将收集的细胞悬液以300×g离心5分钟,弃去上清液。
    22. 将1ml培养基(见食谱)加入细胞沉淀中,并保持在冰上
    23. 重复步骤B18-B22,总共八次。
    24. 八次搅动后,将所有细胞悬浮液(1 ml x 8)合并到一个新的50ml管中
    25. 以300×g离心该细胞悬浮液5分钟,离心后丢弃上清液。
    26. 将细胞沉淀悬浮在1ml RBC裂解缓冲液中,并在室温下孵育1分钟
    27. 将9 ml培养液加入细胞悬浮液中
    28. 以300×g离心细胞悬浮液5分钟,离心后弃去上清液。
    29. 将细胞沉淀悬浮在10ml的培养基中
    30. 将该细胞悬浮液在10厘米未经处理的培养皿上铺板,并在37℃下在5%CO 2培养箱中孵育过夜。
    31. 孵育过夜后,弃去培养基,加入10ml新鲜培养基
    32. 培养分离的心脏细胞超过6天。
      注意:肌成纤维细胞附着在板上;通过改变培养基来除去包括造血细胞在内的未附着的污染细胞。当肌纤维母细胞达到融合前,通过它们。
    33. 在进行体外吞噬试验前一天,从培养皿中弃去培养基,并用10ml PBS洗两次。
    34. 加入1ml胰蛋白酶/ EDTA,37℃孵育1分钟
    35. 加入9ml培养基,并将细胞悬浮液转移到15-ml管中
    36. 以300×g离心细胞悬浮液5分钟,离心后弃去上清液。
    37. 加入10ml的培养基,悬浮细胞沉淀
    38. 计数细胞数,并通过添加培养基将其调整为1×10 5个细胞/ ml。
    39. 将200μl该细胞悬浮液加入到涂有聚-L-赖氨酸(2×10 4个/孔)的8孔载玻片室中,参见Recipes)。
    40. 在5%CO 2培养箱中于37℃孵育过夜。

  3. 坏死性L929细胞的制备
    1. 在含有培养基的10cm未经处理的培养皿上种子2.5×10 6 L929细胞。
    2. 在5%CO 2培养箱中于37℃孵育过夜。
    3. 吸入培养基,并使用10ml无血清DMEM洗涤两次。
    4. 加入10ml含有10μl10mM CMFDA染料的无血清DMEM(见配方)(最终浓度为10μM)。
    5. 在5%CO 2 /培养箱中37℃孵育30分钟
    6. 吸入培养基,并在冰上孵育1分钟
    7. 使用10ml的培养基洗涤4次。
    8. 加入10ml含有20μl10mM Z-VAD-FMK的培养基(参见食谱)(终浓度为20μM)。
    9. 在CO 2 孵育箱中孵育2小时。
    10. 加入10μl10μg/ ml hTNF-α溶液(见配方)(终浓度为10 ng / ml)。
    11. 在5%CO 2培养箱中于37℃孵育4小时。
    12. 将上清液转移到50ml管中 注意:上清液含有坏死性L929细胞。
    13. 以300×g离心细胞悬浮液5分钟,离心后弃去上清液。
    14. 加入10ml的培养基
    15. 完全重复步骤C13和C14三次。
    16. 计数单元格编号。
    17. 用培养基将细胞数调整至1×10 6细胞/ ml
  4. 吞噬试验
    1. 在37℃的5%CO 2培养箱的8孔载玻片室中准备用200μl培养基每孔培养的心肌成纤维细胞(该载玻片在B40中制备) >
    2. 丢弃上清液。
    3. 加入浓度为1×10 6细胞/ ml(2×10 5个细胞/孔)的200μlCMFDA标记的神经细胞L929细胞。
      注意:吞噬的坏死细胞数量是每孔吞噬细胞数量的10倍。
    4. 在37℃,5%CO 2培养箱中孵育3小时。
    5. 弃去上清液,用200μlPBS洗3次 注意:未吞噬的坏死细胞附着于吞噬细胞,通过用PBS洗涤除去这些细胞。
    6. 为了固定细胞,加入200μl的1%PFA / PBS溶液,并在室温下孵育15分钟。
    7. 弃去上清液,用200μlPBS洗涤
    8. 弃去上清液,并从玻璃载玻片上取下室壁。
    9. 用含有0.1%DAPI的FluorSave TM 试剂安装载玻片,并在玻璃载玻片上放置盖玻璃。
    10. 在相位和荧光显微镜下观察 注意:BZ-9000以20倍的倍率捕获图像。获取相位对比图像以及使用GFP-BP和DAPI-BP滤镜捕获的图像。

数据分析

  1. 从12-15个随机选择的字段中捕获三种类型的图像(相位图像以及使用GFP-BP和DAPI-BP滤镜获取的图像)(图1)。
  2. 使用BZ-II图像分析应用程序合并图像。
  3. 计数吞噬细胞和吞噬细胞(CMFDA阳性和合并吞噬细胞)的数量。
    注意:在相位对比图像中,可以以光相和模糊图像的形式观察未受影响的细胞,而吞噬细胞可以以暗相图像的形式观察。不要将这些光相细胞视为吞噬细胞。
  4. 计算吞噬指数(图1) 注意:吞噬指数定义为每个吞噬细胞吞噬细胞的数量。首先,计数由DAPI(粉红色箭头)染色的肌成纤维细胞的核数,然后计数用CMFDA(黄色箭头)标记的被吞噬的坏死细胞数。计数后,吞噬指数计算为吞噬的尸检细胞数除以肌成纤维细胞核数。例如,当肌成纤维细胞核数为11,吞噬细胞数为5时,吞噬指数计算为5/11 = 0.45。计算后,吞噬指数在12-15个随机选择的字段中平均。


    图1.心肌肌成纤维细胞吞噬坏死细胞。手术后3天,从MI小鼠模型中分离出心肌肌成纤维细胞。坏死性L929细胞用CMFDA(绿色)标记。箭头的图片被重新缩放,以便更好地显示吞噬作用。计数吞噬细胞(粉红色箭头)和吞噬细胞(黄色箭头)的数量,并计算吞噬指数。刻度棒=50μm。

食谱

  1. 胶原酶A溶液
    1. 取50ml PBS加至50ml管中
    2. 添加:
      50毫克BSA
      50毫克胰蛋白酶
      50毫克胶原酶A
    3. 混匀,室温下孵育
    4. 通过0.22μmMinisart ®过滤器
      过滤溶液
  2. 培养基
    1. 从500毫升无血清DMEM中除去55毫升无血清DMEM
    2. 加入50ml FBS
    3. 加入5ml青霉素 - 链霉素
  3. 10 mM CMFDA染料
    加入10.76μl的DMSO至50μg的CMFDA染料
  4. 10 mM Z-VAD-FMK
    加入240微升DMSO至1.1毫克Z-VAD-FMK
  5. 10μg/ ml hTNF-α溶液
    1. 将10ml无菌蒸馏水加入到15 ml管中
    2. 加入10毫克BSA
    3. 通过0.22μmMinisart过滤器过滤溶液,制成0.1%BSA溶液
    4. 加入100μl0.1%BSA溶液至10μghTNF-α,制成100μg/ ml hTNF-α溶液,
    5. 向100μl/ ml的hTNF-α溶液中加入0.1μl的BSA溶液90μl
  6. 涂有聚-L-赖氨酸的8孔载玻片室
    1. 加入200μl聚-L-赖氨酸溶液到8孔幻灯片室
    2. 在CO 2 孵育器中孵育3小时
    3. 丢弃聚-L-赖氨酸溶液
    4. 使用PBS清洗

致谢

使用此协议时,请参阅M Nakaya等人。(2017)J Clin Invest。资助由日本教育,文化,体育,科学和技术部(MEXT)[M.N(17H03984)]提供;来自MEXT [到M.N(17H05510)]的创新领域科学研究资助(各类细胞死亡的稳态调节)]。所有动物实验均按照九州大学的指导方针进行。

参考

  1. Horii,Y.,Matsuda,S.,Watari,K.,Nagasaka,A.,Kurose,H。和Nakaya,M。(2017)。< a class =ke-insertfile"href =http: /www.bio-protocol.org/e2553target ="_ blank>确定心脏巨噬细胞和心肌成纤维细胞对凋亡细胞的吞噬作用的测定。生物原型7(18): e2553。
  2. Nakaya,M.,Watari,K.,Tajima,M.,Nakaya,T.,Matsuda,S.,Ohara,H.,Nishihara,H.,Yamaguchi,H.,Hashimoto,A.,Nishida, Nagasaka,A.,Horii,Y.,Ono,H.,Iribe,G.,Inoue,R.,Tsuda,M.,Inoue,K.,Tanaka,A.,Kuroda,M.,Nagata, Kurose,H。(2017)。心肌肌成纤维细胞吞噬死亡细胞有助于心肌梗死后恢复。 J Clin Invest 127(1):383-401。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
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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