Mice.

70 % ethanol in distilled H₂O.

Dissection materials: tweezers, scissors, and sterile scalpel blades.

Conical sterile polypropylene centrifuge tubes (15 mL).

Phosphate-buffered saline (PBS): 1.54 mM KH₂PO₄, 155.17 mM NaCl, and 2.71 mM Na₂HPO₄, pH 7.4, sterilized by autoclaving.

Roswell Park Memorial Institute (RPMI) 1640 medium with antibiotics: 100 U/mL penicillin and 100 μg/mL streptomycin (see Note 1 ).

Cell culture dishes (60 mm and 100 mm).

Paper towels.

Syringes (10 mL) and needles (25G).

Distilled H₂O.

Heat-inactivated (30 min at 56 °C) fetal bovine serum (FBS).

Differentiation medium: RPMI 1640 supplemented with 10 % heat-inactivated FBS, 20 % L-cell conditioned medium (see Note 2 ), 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin.

24-well cell culture plates.

Round coverslips (matching the well size, e.g., 12 mm for 24-well plates).

PBS (as in Subheading 2.1, item 5).

Differentiation medium (as in Subheading 2.1, item 12).

Sterile cell scrapers.

Conical sterile polypropylene centrifuge tubes (50 mL).

Cell counting chamber.

M1 polarization medium: RPMI 1640 supplemented with 10 % heat-inactivated FBS (see Subheading 2.1, item 11), 2 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 ng/mL lipopolysaccharide (LPS), and 10 ng/mL interferon γ (INFγ).

M2 polarization medium: RPMI 1640 supplemented with 10 % heat-inactivated FBS (see Subheading 2.1, item 11), 2 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 ng/mL interleukin 4 (IL-4).

Differentiation medium (as in Subheading 2.1, item 12).

PBS (see Subheading 2.1, item 5).

Permanent marker.

PBS (see Subheading 2.1, item 5).

Starvation medium: RPMI 1640 medium containing 0.1 % heat-inactivated FBS (as in Subheading 2.1, item 11), 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Sheep red blood cells (RBCs) in Alsever’s solution (see Note 3 ) (e.g., Cat. No. SR0053, Oxoid).

Polypropylene microcentrifuge tubes (1.5 mL).

20 mM glucose in HBSS (see Note 4 ).

Rabbit IgM fraction anti-sheep RBCs (e.g., Cat. No. CL9000-M, Cedarlane) (see Note 5 ).

Rabbit IgG fraction anti-sheep RBCs (e.g., Cat. No. 55806, Cappel) (see Note 5 ).

Complement component 5 (C5)-deficient serum (e.g., Cat. No. C1163, Sigma) (see Note 6 ).

Conical sterile polypropylene centrifuge tubes (50 mL).

Ice.

PBS (as in Subheading 2.1, item 5).

4 % formaldehyde in PBS (for 500 mL: in a fume hood, heat 400 mL of PBS to aprox. 60 °C on a hot plate with magnetic stirrer, add 20 g of paraformaldehyde and incubate with mixing, add dropwise 1 M NaOH until all powder is dissolved, fill up with PBS up to 500 mL, cool the solution, filter and adjust pH to 7.4, and use fresh or freeze at −20 °C).

0.5 % (v/v) Triton X-100 in PBS.

0.1 % (v/v) Triton X-100 in PBS.

Blocking solution: PBS supplemented with 1 % (w/v) bovine serum albumin (BSA) and 0.1 % (v/v) Triton X-100.

Anti-rabbit secondary antibody conjugated with a fluorochrome of choice (e.g., FITC, AlexaFluor488, etc.).

Mounting medium of choice containing diamidino-2-phenylindole (DAPI; e.g., ProLong Gold Antifade Mountant with DAPI, Molecular Probes; see Note 7 ).

Microscope glass slides (e.g., 76 × 26 mm).

Fluorescence or confocal microscope.

Euthanize a mouse, from which the lower limbs will be obtained (see Note 8 ).

Spray the mouse with 70 % ethanol to disinfect.

Using tweezers and scissors, remove the skin from both legs (see Note 9 ).

Cut away each leg at the hip joint (where the head of the femur meets the pelvis) and place them in a 15 mL tube containing PBS.

In a flow cabinet, transfer the legs to a paper towel (see Note 10 ) and remove muscles with a sterile blade to obtain clean femurs and tibias.

Put the bones on a 60 mm dish containing RPMI medium with antibiotics.

Hold one bone with sterile tweezers and cut both joints with sterile scissors to gain access to the bone marrow (see Note 11 ).

Aspirate RPMI medium with antibiotics into a 10 mL syringe and fit a 25G needle.

Introduce the needle into the bone lumen and flush out the bone marrow into a new 60 mm dish until the bone color turns white.

Aspirate the medium containing the bone marrow using the same syringe and pass through the needle to disaggregate cells.

Repeat steps 7–10 for all the bones.

Discard the supernatant and add 1 mL of distilled H₂O to lyse RBCs. Pipette up and down for 10 s and add PBS to 15 mL.

Discard the supernatant and resuspend the pellet in 45 mL differentiation medium (see Note 12 ).