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Jan 2017

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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   

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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), Myofibroblast (肌成纤维细胞), Engulfment (吞噬), Apoptosis (细胞凋亡), Myocardial infarction (心肌梗死)

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

Procedures A and B are also applicable to phagocytosis assay of necroptotic cells by 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.
      Note: Male mice are chosen to be subjected to MI operation to avoid the risk of acquiring variable data due to estrous cycle of female mice.
    2. To anesthetize the mouse, administer pentobarbital (50 mg pentobarbital/kg body weight of mouse) (see Recipes) via an intraperitoneal injection.
    3. Fix the mouse on its back on the heating pad with surgical tape.
    4. Perform artificial respiration 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 permanent occlusion of the left coronary artery using 8-0 braided silk (see Video 1).
    7. Close the chest using 5-0 braided silk.
    8. After the operation, give the mouse 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 pentobarbital/kg body weight of mouse) via an intraperitoneal injection.
    2. Open the chest 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 on a clean bench.
      Note: From this step, the experiment should be performed on a clean bench.
    9. Cut each heart into 15 small pieces (2-3 mm2) using two surgical lancets, while keeping them on ice.
    10. Discard PBS using a pipette.
      Note: Please be careful not to discard heart pieces.
    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 the 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. Shake 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 cell suspension, 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 the 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 the 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, aspirate 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.
      Note: Approximately 2.5 x 105 myofibroblasts can be obtained from one mouse.
    39. Add 200 µl of this cell suspension to an 8-well slide chamber coated with poly-L-lysine (2 x 104 cells/well).
    40. Incubate in a 5% CO2 incubator at 37 °C overnight.

  3. Isolation of cardiac macrophages from the MI mouse models
    1. Collect the hearts from the MI mouse models after 3 days of the operation and cut the hearts into small pieces.
      Note: This procedure is the same as that described in steps B1-B10. The experiment should be performed using a clean bench from this step.
    2. Add 10 ml of dispase solution (see Recipes).
    3. Incubate the mixture of the minced hearts and dispase solution in a water bath at 37 °C for 10 min, while shaking at 180 rpm.
    4. Pass the cell suspension through a 70 µm EASYstrainerTM and collect it in a new 50 ml tube.
    5. Centrifuge the cell suspension at 300 x g for 3 min at room temperature and remove it after centrifugation.
    6. Suspend the cell pellet in 2 ml of the culture medium.
    7. Repeat steps C2-C6 thrice.
    8. Combine the three cell suspensions (2 ml x 3) in a 50 ml tube.
    9. Centrifuge the cell suspension at 300 x g for 3 min and discard the supernatant after centrifugation.
    10. Suspend the cell pellet in 1 ml of Red blood cell lysis buffer and incubate for 1 min at room temperature.
    11. Add 9 ml of FACS buffer (see Recipes).
    12. Centrifuge the cell suspension at 300 x g for 3 min at room temperature and discard the supernatant after centrifugation.
    13. Add 1 ml of FACS buffer.
    14. Add 1 µl of the Fixable Viability Dye eFluorTM 780 and incubate on ice for 30 min.
    15. Centrifuge the cell suspension at 300 x g for 3 min at 4 °C and discard the supernatant after centrifugation.
    16. Add 100 µl of primary antibodies (see Recipes) diluted with FACS buffer and incubate on ice for 30 min in a dark environment.
    17. Add 300 µl of FACS buffer.
    18. Centrifuge the cell suspension at 300 x g for 5 min at 4 °C and discard the supernatant after centrifugation.
    19. Add 400 µl of FACS buffer.
    20. Centrifuge the cell suspension at 300 x g for 5 min at 4 °C and discard the supernatant after centrifugation.
    21. Add 400 µl of FACS buffer.
    22. Sort the cardiac macrophages using FACSAria III.
      Note: Cardiac macrophages are identified as Ly6G-/CD11b+/F4/80hi/Ly6Clo (Figure 1).


      Figure 1. The flow gating strategy for sorting of cardiac macrophages. Cardiac cells isolated from the MI mouse models were labeled with eFluorTM 780 viability dye and antibodies against Ly6G, CD11b, F4/80 and Ly6C. First, living cells were gated by eliminating dead cells stained with eFluorTM 780 viability dye. After that, Ly6G-/CD11b+/F4/80hi/Ly6Clo cells (red gate) amongst living cells were sorted as cardiac macrophages by FACSAria III.

    23. Centrifuge the sorted cell suspension at 300 x g for 5 min at 4 °C and discard the supernatant after centrifugation.
    24. Add 1 ml of the culture medium and count the cell number.
      Note: Approximately 1 x 105 macrophages can be obtained from eight mice.
    25. Adjust the cell number to 2.5 x 105 cells/ml by adding the culture medium.
    26. Add 200 µl of this cell suspension to an 8-well slide chamber coated with poly-L-lysine (5 x 104 cells/well, see Recipes).
      Note: Macrophages are smaller than myofibroblasts. To equalize the confluence of macrophages and myofibroblasts, the number of macrophages plated on the 8-well slide chamber is 2.5 times that of the myofibroblasts.
    27. Incubate in a CO2 incubator overnight.

  4. Obtaining apoptotic thymocytes
    1. Euthanize two 4- to 8-week-old female mice by administering sevoflurane.
      Note: Usually, thymocytes of female mice are used for preparation of apoptotic cells owing to the following two reasons: (1) A large number of thymocytes can be obtained because the size of female thymus is bigger than that of male thymus. (2) Most thymocytes are small and uniform in size, whereas other cells such as splenocytes vary in size. Small apoptotic cells are more prone to be engulfed and the uniformity of the cell size aids in ensuring reproducibility of the experimental conditions of phagocytosis.
    2. Open the chest of mice by using sterilized scissors and remove the thymus using sterilized tweezers.
    3. Transfer the thymus into a 6 cm dish containing 4 ml of serum-free DMEM on ice.
    4. Smash the thymus using sterilized, frosted glass slides and suspend the smashed thymus in 4 ml of serum-free DMEM.
    5. Pass the thymocyte suspension through a 40 µm EASYstrainerTM into a 50 ml tube.
    6. Centrifuge the cell suspension at 300 x g for 5 min at room temperature and discard the supernatant after centrifugation.
    7. Suspend the cell pellet in 1 ml of RBC lysis buffer and incubate for 1 min at room temperature.
    8. Add 9 ml of serum-free DMEM.
    9. Centrifuge the cell suspension at 300 x g for 5 min at room temperature and discard the supernatant after centrifugation.
    10. Add 10 ml of serum-free DMEM and count the cell number.
    11. Transfer 2 x 108 cells from the cell suspension into a 50 ml tube.
    12. Add serum-free DMEM and adjust the cell number to 1 x 107 cells/ml.
    13. Add 1/10,000 times the volume of the cell suspension of 10 mM CMFDA dye (see Recipes) (final concentration would be 1 µM) and incubate for 30 min at 37 °C in a CO2 incubator, while blocking light by covering with an aluminum foil.
      Note: When the final volume of the cell suspension reaches 20 ml in step D12, add 2 µl of 10 mM CMFDA dye and adjust the final concentration to 1 µM.
    14. Dispense 3 ml of the cell suspension into each 15 ml tube and add 12 ml of the culture medium to each tube.
      Note: To stop CMFDA labeling reaction, use the culture medium containing FBS.
    15. Incubate on ice for 5 min.
    16. Centrifuge the cell suspension at 300 x g for 5 min at room temperature and discard the supernatant after centrifugation.
    17. Add 10 ml of the culture medium and combine all the cell suspensions in one 15 ml tube.
    18. Repeat steps D16-D17 thrice.
    19. Count the cell number.
    20. Adjust the cell number to 1.2 x 107 cells/ml in one 15 ml tube.
    21. Add 1/1,000 times the volume of the cell suspension of 10 mM dexamethasone (see Recipes) (final concentration would be 10 µM).
      Note: When final volume of the cell suspension reaches 10 ml in step D20, add 10 µl of 10 mM dexamethasone and adjust the final concentration to 10 µM.
    22. Incubate the cell suspension for 5 h at 37 °C in a CO2 incubator, while blocking light by covering with an aluminum foil.
    23. Centrifuge the cell suspension at 300 x g for 5 min at room temperature and discard the supernatant after centrifugation.
    24. Add 10 ml of the culture medium.
    25. Repeat steps D23-D24 thrice.
    26. Count the cell number and adjust it to 1 x 106 cells/ml (for cardiac myofibroblasts) and 2.5 x 106 cells/ml (for cardiac macrophages).
      Note: Approximately 2.5 x 108 apoptotic thymocytes can be obtained from one mouse.

  5. In vitro phagocytosis assay
    1. Prepare the cardiac myofibroblasts and cardiac macrophages cultured on the 8-well slide chamber.
    2. Discard the medium.
    3. Add 200 µl of apoptotic thymocytes (2 x 105 cells/well for myofibroblasts and 5 x 105 cells/well for macrophages).
      Note: The number of engulfed apoptotic cells is ten times the number of phagocytes per well.
    4. Incubate for 3 h at 37 °C in a CO2 incubator.
    5. Discard the supernatant and wash with 200 µl of PBS thrice by using a pipette.
      Note: Unengulfed apoptotic 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. Manually count the number of 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 (Figure 2).
    Note: Phagocytosis index is defined as the number of engulfed cells per phagocyte. First, count the number of nuclei of myofibroblasts or macrophages that are stained by DAPI (pink arrowheads) and then count the number of engulfed apoptotic cells labeled with CMFDA (yellow arrowheads). After the counting, phagocytosis index is calculated as the number of engulfed apoptotic cells divided by the number of nuclei of myofibroblasts or macrophages. For example, when the number of nuclei of myofibroblasts is 22 and the number of engulfed apoptotic thymocytes is 7, phagocytosis index is calculated as 7/22 = 0.32. Following the calculation, phagocytosis index is averaged over 12-15 randomly selected fields.


    Figure 2. Cardiac myofibroblasts and macrophages engulf the apoptotic thymocytes. Cardiac myofibroblasts and macrophages were isolated from the MI mouse models 3 days after operation. The mouse apoptotic thymocytes 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.

Notes

In our opinion, this protocol is applicable for measuring phagocytosis by other cell types as well. In fact, we measured phagocytosis of apoptotic thymocytes by NIH3T3 cells using the same protocol (Nakaya et al., 2006).
It should be noted that the cell sizes of phagocytes and apoptotic cells have to be considered in phagocytosis assay. The size of apoptotic cells should be smaller than that of phagocytes.

Recipes

  1. Pentobarbital (prepared fresh)
    1. Transfer 1 ml of pentobarbital from vial into a 15-ml tube
    2. Add 12 ml of sterile distilled water
  2. Collagenase A solution (prepared fresh)
    1. Take 50 ml in 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
  3. Culture medium (can be kept for 1 month at 4 °C)
    1. Remove 55 ml of serum-free DMEM from 500 ml of serum-free DMEM
    2. Add 50 ml of heat-inactivated FBS (30 min at 56 °C)
    3. Add 5 ml of penicillin-streptomycin
  4. Dispase solution (50 ml) (prepared fresh)
    1. Take 125 mg of trypsin in a 50-ml tube
    2. Add:
      112 mg of dispase
      100 µl of 0.5 M EDTA·2Na
      Add PBS up to 50 ml
    3. Mix well and filter the solution through a 0.22-µm Minisart® filter
  5. FACS buffer (prepared fresh)
    PBS supplemented with 2% FBS
  6. Primary antibodies/FACS buffer (prepared fresh)
    1. Take 100 µl of FACS buffer in a 1.5-ml tube
    2. Add:
      1 µl of FITC-conjugated anti-Ly6C antibody (used at a dilution of 1:100)
      0.5 µl of PE-conjugated anti-Ly6G antibody (used at a dilution of 1:200)
      1 µl of APC-conjugated anti-F4/80 antibody (used at a dilution of 1:100)
      0.5 µl of PerCP-Cy5.5-conjugated anti-CD11b antibody (used at a dilution of 1:200)
    3. Mix well
    4. Incubate on ice
  7. 8-well slide chamber coated with poly-L-lysine (prepared fresh)
    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 once
  8. 10 mM CMFDA dye (can be kept for several months at -20 °C)
    Add 10.76 µl of DMSO to 50 µg of CMFDA dye
  9. 10 mM Dexamethasone (can be kept for several months at -20 °C)
    Add 50 µl of DMSO to 164.5 µg of Dexamethasone

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). Phagocytosis assay of necroptotic cells by cardiac myofibroblasts. Bio Protoc 7(18): e2252.
  2. Nakaya, M., Tanaka, M., Okabe, Y., Hanayama, R. and Nagata, S. (2006). Opposite effects of rho family GTPases on engulfment of apoptotic cells by macrophages. J Biol Chem 281(13): 8836-42.
  3. 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,许多心肌细胞凋亡。 这些凋亡心肌细胞被吞噬细胞迅速吞噬。 如果死细胞没有被吞没,其有害物质被释放到外面,导致炎症的诱导。 因此,需要去除这些死细胞。 然而,每个吞噬细胞类型对梗塞心脏中凋亡细胞的去除的贡献仍未解决。 在这里,我们描述了吞噬作用测定的体外方案来比较心脏巨噬细胞和心脏肌成纤维细胞的吞噬能力。
【背景】长期以来一直认为,心脏巨噬细胞消除了失败的心脏中产生的凋亡细胞。 然而,我们发现负责组织纤维化的心脏肌成纤维细胞也具有在MI后吞噬凋亡细胞的能力(Nakaya等,2017)。 这一发现促使我们比较心脏巨噬细胞和心脏肌成纤维细胞的吞噬能力。 在这里,我们提供了一个详细的体外吞噬试验方案来评估吞噬吞噬的程度。

关键字:吞噬分析, 肌成纤维细胞, 吞噬, 细胞凋亡, 心肌梗死

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. 磨砂玻璃片(松本玻璃,目录号:S2112)
  18. 盖玻璃(松本玻璃,目录号:C024601)
  19. 40μmEASYstrainer TM (Greiner Bio One International,目录号:542040)
  20. 铝箔
  21. 1.5 ml管(BMBio,目录号:BM-15)
  22. 0.22μmMinisart ®过滤器(Sartorius,目录号:16534-K)
  23. 野生型C57BL / 6JJmsSlc小鼠(日本SLC)
  24. 戊巴比妥(Somnopentyl)(Kyoritsu Seiyaku,目录号:SOM02-YA1312)
  25. 水(NACALAI TESQUE,目录号:06442-95)
  26. 磷酸盐缓冲盐水(PBS)(NACALAI TESQUE,目录号:14249-95)
  27. 红细胞(RBC)裂解缓冲液(Roche Diagnostics,目录号:11814389001)
  28. 胰蛋白酶/乙二胺四乙酸(EDTA)(NACALAI TESQUE,目录号:35554-64)
  29. 可固定的可行性染料eFluor TM(Thermo Fisher Scientific,eBioscience TM,目录号:65-0865-14)
  30. 七氟烷(和光纯药,目录号:193-17791)
  31. 多聚甲醛(PFA)(NACALAI TESQUE,目录号:26126-25)
  32. 4',6-二脒基-2-苯基吲哚(DAPI)(Dojindo,目录号:340-07971)
  33. FluorSave TM (默克,目录号:345789)
  34. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153-100G)
  35. 胰蛋白酶(Sigma-Aldrich,目录号:T4799-5G)
  36. 胶原酶A(Roche Diagnostics,目录号:10103586001)
  37. 无血清DMEM(NACALAI TESQUE,目录号:08458-16)
  38. 致命的牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10437028)
  39. 青霉素 - 链霉素(NACALAI TESQUE,目录号:09367-34)
  40. Dispase(Roche Diagnostics,目录号:04942078001)
  41. EDTA•2Na(Dojindo,目录号:345-01865)
  42. FITC结合的抗Ly6C抗体(BioLegend,目录号:128006)
  43. PE结合的抗Ly6G抗体(BioLegend,目录号:127607)
  44. APC结合的抗F4 / 80抗体(BioLegend,目录号:123116)
  45. PerCP / Cy5.5结合的抗CD11b抗体(BioLegend,目录号:101230)
  46. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2650)
  47. 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)
  48. 聚-L-赖氨酸溶液(Sigma-Aldrich,目录号:P4707)
  49. 地塞米松(Sigma-Aldrich,目录号:D1756-25MG)
  50. 胶原酶A溶液(参见食谱)
  51. 培养基(见食谱)
  52. Dispase解决方案(请参阅配方)
  53. FACS缓冲区(见配方)
  54. 主要抗体(见食谱)
  55. 涂有聚-L-赖氨酸的8孔滑动室(参见食谱)
  56. 10 mM CMFDA染料(见食谱)
  57. 10 mM地塞米松(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. 手术工具如镊子和小剪刀(工具可以从NATSUME SEISAKUSHO和MEISTER购买)
  8. 洁净台(Panasonic Healthcare,型号:MCV-B131S)
  9. 水浴(TAITEC,型号:Personal-11)
  10. 离心机(TOMY SEIKO,型号:LC-200)
  11. CO 2培养箱(SANYO,型号:MCO-18AIC)
  12. 细胞分选仪(BD,BD Bioscience,型号:FACSARIA III)
  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也适用于心肌成纤维细胞(Horii等人,2017年)对坏死细胞的吞噬作用测定。

  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。
      注意:选择雄性小鼠进行MI手术,以避免由于雌性小鼠的发情周期而获得可变数据的风险。
    2. 要麻醉小鼠,通过腹膜内注射施用戊巴比妥(50mg戊巴比妥/ kg体重的小鼠)(参见食谱)。
    3. 用手术胶带将鼠标固定在加热垫上。
    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编织丝执行左冠状动脉永久性闭塞(见视频1)。
    7. 使用5-0编织丝来关闭胸部。
    8. 手术后,给予适当的治疗,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.


  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戊巴比妥/ kg体重的小鼠),在MI手术3天后对两只小鼠进行安乐死。
    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. 收集心脏并清除心房。
    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6厘米盘中 注意:从这一步,实验应该在干净的工作台上进行。
    9. 使用两个外科手术刀将每个心脏切成15个小块(2-3 mm 2 ),同时保持在冰上。
    10. 使用移液器丢弃PBS。
      注意:请注意不要丢弃心脏碎片。
    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管中并清除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溶液加入到管中,该溶液现在含有残留物质
    19. 摇动管,含有切碎的心脏和胶原酶A溶液的混合物,在水浴中以120rpm和37℃10分钟。
    20. 将含有分离细胞的细胞悬浮液通过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分钟并弃去上清液。
    22. 将1ml培养基(见食谱)加入细胞沉淀并保存在冰上。
    23. 总共重复步骤B18-B22八次。
    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离心细胞悬浮液5分钟,离心后弃去上清液。
    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厘米未处理的培养皿上铺板,并在37℃的5%CO 2培养箱中孵育过夜。
    31. 孵育过夜后,吸取培养基,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,从培养皿中丢弃培养基,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9 ml培养液,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的培养基并悬浮细胞沉淀。
    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。
      注意:可以从一只老鼠获得约2.5×10 5
    39. 将200μl该细胞悬浮液加入涂有聚-L-赖氨酸(2×10 4个细胞/孔)的8孔载玻片室中。
    40. 在5%CO 2培养箱中于37℃孵育过夜。

  3. 从MI小鼠模型中分离心脏巨噬细胞
    1. 3天后,从MI小鼠模型收集心脏,将心脏切成小块 注意:此步骤与步骤B1-B10中所述的相同。实验应使用本步骤中的干净工作台进行。
    2. 加入10ml分散液(参见食谱)。
    3. 将切碎的心脏和分液溶液的混合物在37℃的水浴中孵育10分钟,同时以180rpm摇动。
    4. 将细胞悬浮液通过70微米EASYstrainer TM ,并将其收集在新的50ml管中。
    5. 在室温下以300×g离心细胞悬浮液3分钟,离心后除去。
    6. 将细胞沉淀物悬浮于2ml培养基中
    7. 重复步骤C2-C6三次。
    8. 将三种细胞悬浮液(2ml×3)混合在50ml管中
    9. 以300×g离心细胞悬浮液3分钟,离心后弃去上清液。
    10. 将细胞沉淀悬浮在1ml红细胞裂解缓冲液中,并在室温下孵育1分钟
    11. 加入9 ml FACS缓冲液(参见食谱)
    12. 在室温下以300×g离心细胞悬浮液3分钟,离心后弃去上清液。
    13. 加入1ml FACS缓冲液。
    14. 加入1μlFixable Viability Dye eFluor< sup> 780,并在冰上孵育30分钟。
    15. 在4℃将细胞悬浮液以300×g离心3分钟,离心后弃去上清液。
    16. 加入100μl用FACS缓冲液稀释的一抗(参见食谱),并在黑暗环境中在冰上孵育30分钟。
    17. 加入300μlFACS缓冲液。
    18. 在4℃下以300×g离心细胞悬浮液5分钟,离心后弃去上清液。
    19. 加入400μlFACS缓冲液。
    20. 在4℃下以300×g离心细胞悬浮液5分钟,离心后弃去上清液。
    21. 加入400μlFACS缓冲液。
    22. 使用FACSAria III对心脏巨噬细胞进行分类。
      注意:心脏巨噬细胞被识别为Ly6G - / CD11b + 功能 / F4 / 80 喜 /的Ly6C LO (图1)。


      图1.用于分选心脏巨噬细胞的流动门控策略。从MI小鼠模型分离的心脏细胞用eFluor TM 780活力染料和抗Ly6G,CD11b, F4 / 80和Ly6C。首先,通过消除用eFluor TM 780活性染料染色的死细胞门活细胞。之后,Ly6G - / CD11b + / F4 / 80 喜 /的Ly6C LO 活细胞中的细胞(红门)被FACSAria III分类为心脏巨噬细胞。

    23. 在4℃下以300×g离心分选的细胞悬浮液5分钟,离心后弃去上清液。
    24. 加入1 ml培养基,计数细胞数。
      注意:可从八只小鼠获得大约1 x 10 5 巨噬细胞。
    25. 通过添加培养基将细胞数调整至2.5×10 5个细胞/ ml
    26. 将200μl该细胞悬浮液加入涂有聚-L-赖氨酸(5×10 4个细胞/孔的8孔)载玻片室中,参见食谱。
      注意:巨噬细胞比肌成纤维细胞小。为了均衡巨噬细胞和肌成纤维细胞的融合,在8孔载玻片室上铺设的巨噬细胞的数量是肌成纤维细胞的2.5倍。
    27. 在一个CO 2 孵化器过夜。

  4. 获得凋亡的胸腺细胞
    1. 通过施用七氟醚来安乐死2至4周龄的雌性小鼠 注意:通常,雌性小鼠的胸腺细胞由于以下两个原因用于制备凋亡细胞:(1)由于雌性胸腺的大小大于雄性胸腺的大小,因此可以获得大量的胸腺细胞。 (2)大多数胸腺细胞大小均匀,而其他细胞如脾细胞大小不等。小细胞凋亡细胞更容易被吞噬,细胞大小的均匀性有助于确保吞噬实验条件的重复性。
    2. 用无菌剪刀打开小鼠胸部,用无菌镊子去除胸腺
    3. 将胸腺转移到含有4毫升无血清DMEM的6厘米盘中
    4. 使用无菌磨砂玻璃片将胸腺粉碎,并将破碎的胸腺悬浮在4毫升无血清DMEM中。
    5. 将胸腺细胞悬浮液通过40μmEASYstrainer TM 放入50ml管中。
    6. 在室温下以300×g离心细胞悬浮液5分钟,离心后弃去上清液。
    7. 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
    8. 加入9毫升无血清的DMEM
    9. 在室温下以300×g离心细胞悬浮液5分钟,离心后弃去上清液。
    10. 加入10ml无血清的DMEM并计数细胞数。
    11. 将2×10 8细胞从细胞悬浮液转移到50ml管中
    12. 加入无血清DMEM,并将细胞数调整至1×10 7个细胞/ ml。
    13. 加入10mM CMFDA染料的细胞悬浮液的体积的1/10000倍(参见食谱)(终浓度为1μM),并在37℃下在CO 2培养箱中孵育30分钟,同时用铝箔覆盖遮光 注意:当步骤D12中细胞悬浮液的最终体积达到20ml时,加入2μl10mM CMFDA染料并将终浓度调整至1μM。
    14. 将3ml细胞悬浮液分配到每个15ml管中,并向每个管中加入12ml培养基。
      注意:要停止CMFDA标记反应,请使用含有FBS的培养基。
    15. 在冰上孵育5分钟。
    16. 在室温下以300×g离心细胞悬浮液5分钟,离心后弃去上清液。
    17. 加入10ml的培养基,并将所有细胞悬浮液混合在一个15ml管中
    18. 重复步骤D16-D17三次。
    19. 计数单元格编号。
    20. 在一个15 ml管中将细胞数调整为1.2×10 7细胞/ ml
    21. 加入10mM地塞米松细胞悬浮液体积的1/100倍(参见食谱)(终浓度为10μM)。
      注意:当在步骤D20中细胞悬浮液的最终体积达到10ml时,加入10μl10mM地塞米松并将终浓度调节至10μM。
    22. 在37℃下在CO 2培养箱中孵育细胞悬浮液5小时,同时通过用铝箔覆盖来阻挡光。
    23. 在室温下以300×g离心细胞悬浮液5分钟,离心后弃去上清液。
    24. 加入10ml的培养基
    25. 重复步骤D23-D24三次。
    26. 计数细胞数,并将其调整为1×10 6细胞/ ml(用于心肌成肌纤维细胞)和2.5×10 6细胞/ ml(用于心脏巨噬细胞)。 /> 注意:可以从一只小鼠获得约2.5×10 7细胞凋亡的胸腺细胞。

  5. 吞噬作用测定
    1. 准备在8孔载玻片室培养的心脏肌成纤维细胞和心脏巨噬细胞
    2. 舍弃介质。
    3. 向巨噬细胞中加入200μl凋亡的胸腺细胞(2×10 5个细胞/孔用于肌成纤维细胞和5×10 5个细胞/孔)。
      注意:吞噬凋亡细胞的数量是每孔吞噬细胞数量的十倍。
    4. 在37℃的CO 2培养箱中孵育3小时。
    5. 弃去上清液,用200μlPBS洗3次,用移液管 注意:未吞噬凋亡细胞连接到吞噬细胞,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,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,并将玻璃盖放在载玻片上。
    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. 手动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2) 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或巨噬细胞的核数,然后计数用CMFDA(黄色箭头)标记的被吞噬的凋亡细胞数。计数后,吞噬指数计算为吞噬凋亡细胞数除以肌成纤维细胞或巨噬细胞核的数量。例如,当肌成纤维细胞核数为22,吞噬凋亡胸腺细胞数为7时,吞噬指数计算为7/22 = 0.32。计算后,吞噬指数在12-15个随机选择的字段中平均。


    图2.心肌肌成纤维细胞和巨噬细胞吞噬凋亡性胸腺细胞。手术后3天,从MI小鼠模型中分离出心肌肌成纤维细胞和巨噬细胞。小鼠凋亡胸腺细胞用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。计数吞噬细胞数量(粉红色箭头)和吞噬细胞(黄色箭头),计算吞噬指数。刻度棒=50μm。

笔记

我们认为,该方案适用于测量其他细胞类型的吞噬作用。事实上,我们使用相同的方案测量NIH3T3细胞的凋亡胸腺细胞的吞噬作用(Nakaya等,2006)。
应该注意的是吞噬细胞和细胞凋亡细胞的细胞大小必须在吞噬试验中考虑。凋亡细胞的大小应小于吞噬细胞的大小。

食谱

  1. 戊巴比妥(准备新鲜)
    1. 将1毫升戊巴比妥从小瓶转移到15-ml管中
    2. 加入12毫升无菌蒸馏水
  2. 胶原酶A溶液(新鲜制备)
    1. 取50毫升的PBS到一个50毫升的管子
    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0.22-μmMinisart 过滤器过滤溶液
  3. 培养基(4℃保存1个月)
    1. 从500毫升无血清DMEM中除去55毫升无血清DMEM
    2. 加入50ml热灭活的FBS(56℃30分钟)
    3. 加入5ml青霉素 - 链霉素
  4. 分散液(50ml)(新鲜制备)
    1. 将125毫克胰蛋白酶置于50毫升管中
    2. 添加:
      112毫克dispase
      100μl0.5M EDTA•2Na
      加PBS至50 ml
    3. 混合均匀,并通过0.22-μmMinisart 过滤器过滤溶液
  5. FACS缓冲液(新鲜制备)
    PBS补充2%FBS
  6. 一抗/ FACS缓冲液(新鲜制备)
    1. 将100μlFACS缓冲液置于1.5 ml管中
    2. 添加:
      1μlFITC缀合的抗Ly6C抗体(以1:100的稀释度使用)
      0.5μlPE结合的抗Ly6G抗体(以1:200的稀释度使用)
      1μlAPC-缀合的抗F4 / 80抗体(以1:100的稀释度使用)
      将0.5μlPerCP-Cy5.5缀合的抗CD11b抗体(以1:200稀释使用)
    3. 混合好
    4. 在冰上孵化
  7. 涂有聚-L-赖氨酸的8孔载玻片室(制备新鲜)
    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洗一次
  8. 10 mM CMFDA染料(可在-20°C保存数月)
    加入10.76μl的DMSO至50μg的CMFDA染料
  9. 10 mM地塞米松(可在-20°C保存数月)
    向地塞米松164.5微克加入50微升DMSO

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/e2552"target =“_ blank">心脏肌成纤维细胞对坏死细胞的吞噬作用。生物样品 7(18):e2252。
  2. Nakaya,M.,Tanaka,M.,Okabe,Y.,Hanayama,R。和Nagata,S。(2006)。 rho家族GTPases的相反作用在巨噬细胞吞噬细胞凋亡细胞中的作用。生物化学281(13):8836-42。
  3. 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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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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