Marrowderived macrophages

• 25 cm2 and 75 cm2 tissue culture flasks (Falcon)

• Sterile rubber policeman

• Pasteur pipette

Protocol 9 continued

• Pathogen-free mice

• 14,1% Nycoprep (Accurate Chemical)

• Dulbecco's modified Eagle medium (DMEM) (Gibco BRL)

• Macrophage colony-stimulating factor {M-CSF) (Genzyme)

Method

1 Extrude bone marrow plugs as described in Protocol 8, using PBS as the flushing solution,

2 Pool all extruded material into a 50 ml polypropylene tube and resuspend gently using a Pasteur pipette.

3 Centrifuge at 500 g for 10 min at room temperature,

4 Discard supernatant fluids.

5 Resuspend the pelleted cells in 5 ml serum-free DMEM.

6 Place 5 ml of 14.1% Nycoprep in a 15 ml conical tube.

7 Overlay with the 5 mi solution of bone marrow cells and centrifuge at 500 g for 20 min at room temperature with no brake.

8 Remove the cells at the interface with a Pasteur pipette,

9 Transfer to a fresh 15 ml tube and centrifuge at 500 g for 10 min at 4°C.

10 Resnspend the cell pellet in complete DMEM to a concentration of 5 x 106 cells/ml,

11 Add 1 x 107 cells to individual 25 cin3 tissue culture flasks in 10 ml complete DMEM.

12 Incubate flasks for 24 h at 37°C in a 5% COj incubator.

13 Harvest the non-adherent cells and transfer to a 75 cm3 tissue culture flask,

14 Add 10 ml complete DMEM containing M-CSF at a final concentration of 500-1000 U/ml.

16 Add an additional 10 ml complete DMEM with M-CSF (500-1000 U/ml) to each flask,

17 Incubate cells for an additional three days.

18 At the end of seven days, remove or decant culture medium.

19 Wash the adherent cells with 10 ml PBS.

20 Add 5 ml of filter sterilized dispase solution (pre-warmed to 37 °C),

21 Incubate at 37°C for 5 min.

22 Rap the flask sharply against the palm of the hand to dislodge adherent cells.

23 Remove the cells from the bottom of the flask by scraping gently with a rubber policeman.

• Complete DMEM: medium plus 10% FCS, 2 mM glutamine, 15 mM Hepes buffer, 0.02% sodium bicarbonate, 100 U/ml penicillin, 100 ^g/ml streptomycin {Gibco BRL)

Protocol 9 continued

24 Add 10 ml complete DMEM to the flask.

25 Resuspend the cells and place them into a 50 ml tube.

26 Centrifuge the cells 500 g for 10 min at 4 DC.

27 Resuspend the cells in 5 ml complete DMEM.

The yield of bone marrow-derived macrophages after seven days of culture should approach 2-3 x 10s macrophages per 10 x 10fi cultured bone marrow cells. Other growth factors such as granulocyte-macrophage colony-stimulating factor or interleukin-3 can be used in place of M-CSF. Using these growth factors will result in a yield of 1 x 106 macrophages per 10 x 106 bone marrow cells (16).

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