Isolation of resident bone marrow macrophages

Equipment and reagents

• 30 ml syringes and 25-gauge needles

• 50 ml polypropylene tubes

• Sterile circular coverslips (PGC Scientific)

• 24-well tissue culture plates (Fisher Scientific)

• Sterile forceps and scissors

• Rubber tubing to fit 30 ml syringe

• Tube rotator/rocker

• Pathogen-free mice

• Flushing solution: RPMI 1640 (Gibco BRL) containing 0.05% collagena.se type 3 {Boehringer Mannheim) and 0.001% DNase type I {Sigma Chemical)

• RPMI 1640 containing 10% endotoxin-free FCS (Gibco BRL)

• RPMI 1640 containing 30% endotoxin-free FCS (Gibco BRL)

• Ficoll-Hypaque (Pharmacia)

Method

1 Sacrifice the mice by cervical dislocation or C02 asphyxiation.

2 Make an incision at the top of each hind leg and pull the skin down towards the foot to expose the muscle,

3 Cut off the hind legs and place them on a sterile 100 x 15 mm Petri dish.

4 Remove the muscle from the bones by cutting with scissors then pulling the muscle downward and away with forceps, and remove the foot and the skin.

5 Cut the tibia from the femur at the joint.

6 Fill a 5 ml syringe with flushing solution and attach a 25-gauge needle.

7 Wash the bone marrow cavity free of cells by inserting the needle and injecting 2-5 ml of flushing solution while holding the bone over the Petri dish at a 45° angle with sterile forceps.

8 Collect the bone marrow plugs in a fresh Petri dish and place it on ice while continuing the procedure with the remaining bones.

9 Suspend the bone marrow plugs collected from one mouse in a 50 ml tube in volume of 10 ml flushing solution and incubate the mixture for 1 h at 37°C, with constant rotation (one revolution per second).

10 Stop the enzymatic digestion of the extruded material by adding FCS to the mixture at a final concentration of 1% (v/v).

11 Pool the digests of material from the bone marrow of two mice.

12 Centrifuge the cells at 100 g for 10 minat room temperature.

13 Gently resu spend the pelleted cells in 3 ml RPMI 1640 without serum.

Protocol 8 continued

14 Place a 3 ml layer of Ficoll-Hypaque in a 20 ml syringe with rubber tubing attached and clamped closed.

15 Carefully layer 20 ml RPMI containing 30% FCS over the Ficoll-Hypaque cushion using a 10 ml pipette.

16 Layer the 3 ml cell suspension over the RPMI/Ficoll-Hypaque using a Pasteur pipette and let the gradient stand for 1 h at room temperature.

17 Unclamp the tubing and collect 2 ml fractions into a 24-well plate.

18 Inspect the fractions for the presence of cell clusters free of contaminating single cells using a phase-contrast microscope.

19 Pool the fractions containing the clusters.

20 To separate the bone marrow clusters, layer 5 ml of the bone marrow digest over 10 ml RPMI containing 30% FCS in a 50 ml conical tube.

21 Let the gradient stand for 1 h at room temperature.

22 Aspirate the upper 14 ml of medium, leaving 1 ml remaining in the tube—this contains the clusters of cells with macrophages.

23 Wash the cells twice with RPMI 1640 alone by centrifugation for 10 minat 100 g.

24 Resuspend the cells in RPMI 1640 plus 10% FCS using 0.8 ml per original column number.

25 Add 100 n-1 of cells to sterile circular coverslips in 24-well plates and allow them to adhere at 37 °C for 30 min in a 5% C02 incubator.

27 Incubate the cells at 37°C in a 5% C02 incubator for 3 h.

28 Rinse the coverslips five times by washing with 1 ml PBS per well and removing the PBS with a Pasteur pipette.

29 Place 1 ml PBS per well and incubate the cultures for 30 min at room temperature.

30 Rinse the coverslips repeatedly with PBS. The remaining adherent cells are 50% bone marrow macrophages.

Protocol 9

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