Macrophage depletion and reconstitution

For macrophage depletion and reconstitution, we referred to the protocol published by Weisser, S.B. et al., (2012). Bone marrow-derived macrophage isolation protocol was modified from a paper published by Trouplin et al., (2013). For in vivo macrophage depletion, the liposome-encapsulated clodronate was administrated according to the manufacturer’s protocol. We wrote down our version of the combined protocol as detailed as possible and are happy to address questions about the procedure should there be anything unclear.

Author: Wei-Jia Luo, Ph.D. Email address: f04424006@ntu.edu.tw

Materials

1. DMEM-10 (DMEM with 10% FBS, 1% L-Glu, 1% APS, sodium pyruvate, HEPES)
2. M-CSF (Peprotech, 315-02)
3. Liposome-encapsulated clodronate (Liposoma)
4. 0.2% BSA in PBS
5. 0.02% EDTA in PBS
6. ACK buffer
7. 10-cm dish (coating and non-coating)
8. 70 μm Nylon cell strainer
9. Sterile scissors and forceps
10. 1 mL sterile syringe with 26 gauge needle

Methods

1. Euthanize a mouse by cervical dislocation. Ensure the use of sterile surgical blades throughout the entire experiment. Sterilize the skin by applying 70% alcohol.
2. Create an incision at the top of each hind leg and pull down the skin towards the foot to expose the underlying muscle.
3. Sever the hind legs and remove the skin using sterile scissors and forceps. Cut off the femur and tibia carefully and remove any remaining flesh and muscle attached to the bones.
4. Place the bones in a sterile Petri dish (35/10 mm) containing 5 ml of 75% alcohol and immerse for 1 minute to kill the remaining muscular cell.
5. Wash the bones with 5 ml of sterile, ice-cold 1x PBS with 0.02% EDTA and immerse the bones into a sterile Petri dish (35/10 mm) containing 5 ml of ice-cold DMEM.
6. Load 600 uL DMEM into a 1 mL sterile syringe with a 26 gauge needle attached to the top of it. Use sterile forceps to hold a bone and scissors to cut both ends of it.
7. Insert the needle into the marrow cavity to flush out bone marrow cells. Perform the step repeatedly until most of the bone marrow cells are flushed out.
8. Pipetting the bone marrow several times with the same syringe to break it up.
9. Collect the supernatant and filter it through a 70 μm Nylon cell strainer to remove solid debris.
10. Transfer the filtrate to a 15 mL tube and centrifuge at 1500 rpm for 5 min at 4 °C.
11. Discard the supernatant and dissociate the cell pellet with 2 mL ACK buffer for 1 minute to lyse the RBCs. Add 12 mL of PBS containing 0.2% BSA and centrifuge the mixture at 1500 rpm for 5 min at 4 °C.
12. Discard the supernatant, dissolve the cell pellet with DMEM-10, and dispense the cell suspension to 2 10-cm culture dishes. Incubate them for 4 hr at 37 °C.
13. Collect the supernatant and centrifuge it at 1500 rpm for 5 min at 4 °C.
14. Discard the supernatant and resuspend cells with 32 mL DMEM-10 containing 10 ng/mL M-CSF.
15. Filter it through a 70 μm Nylon cell strainer and dispense the filtrate to 4 10-cm non-coating dished (8 mL/ dish). Incubate them for 3 days at 37 °C.
16. Add 4 mL DMEM-10 supplemented with 30 ng/mL M-CSF to each dish and mix by shaking gently. Incubate cells for additional 4 days at 37 °C.
17. Remove the supernatant and wash BMDMs 2 times with PBS. Add 5 ml DMEM-10 that has been warmed to 37 °C. Detach BMDMs by gently scraping with a rubber policeman.
18. Collect BMDMs in tubes and centrifuge at 1500 rpm for 5 min. Gently dissociate cell pellets in 10 mL DMEM-10. Count BMDMs in the presence of trypan blue.
19. Dilute the macrophages in sterile PBS to achieve a concentration of 10^7 cells/ml. This concentration enables the intravenous injection of 10^6 macrophages in a total volume of 100 μL.
20. Before macrophage depletion, allow the liposome-encapsulated clodronate (Clo-Lip) and control liposome to acclimate to room temperature for at least 1 hour and mix it evenly prior to every single injection.
21. To deplete macrophages, inject 100 µL of Clo-Lip suspension /10 grams of animal weight via tail intravenous injection. (e.g. inject 200 uL Clo-Lip suspension or control solution into the tail vein of mice weighing 20 grams.)
22. 2 days after the depletion, with a 1 ml syringe equipped with a 26 gauge needle, slowly administer 100 μL of the macrophage suspension generated from step 19 (containing 10^6 macrophages) into the injection site intravenously.
23. 1 day after the transplantation, mice are treated with LPS or PBS intraperitoneally. The serum is collected and IL-12 as well as IFN-g are analyzed 4 and 8 hours after the challenge.