Primary macrophage culture assays

Detailed primary macrophage culture protocol

1. On day 1, inject 3 ml of 4% thioglycollate solution intraperitoneally to wildtype C57BL/6 mice.
2. On day 4 or 5, euthanize the mice (with IACUC-approved protocols specific to your lab) and continue the next steps in a biosafety cabinet under sterile conditions.
3. Use a pair of scissors and forceps to peel the skin from the abdominal region. Fill a 10 ml syringe with 10 ml of ice-cold PBS and fit an 18 gauge needle.
4. Lifting the abdominal wall with the forceps, inject the PBS into the peritoneum. Use the same syringe to draw out the PBS from the peritoneum.
5. Repeat the injection with fresh PBS twice more. When completed, filter the cells with a 0.22 mm filter.
6. Spin down the cells at 1000 rpm for 5 min at 4°C . Aspirate and add 1 ml of ACK buffer (0.15 M NH₄Cl, 1 mM KHCO₃, 0.1 mM EDTA, pH 7.3) and incubate for 3 min on ice.
   Note: If cells from 2-3 mice were combined in a single Corning tube, add 2-3 ml ACK buffer.
7. Once incubation is complete, add PBS up to 50 ml and centrifuge 1000 rpm for 5 min at 4°C.
8. Wash the cells once with 30 ml of PBS and repeat the centrifugation step.
9. Resuspend the cell pellet in a small volume (1 to 5 ml) of 10% FBS-RPMI 1640 containing 1% penicillin/streptomycin and count.
10. Plate the cells at the required density (e.g. 80,000 cells in 1 well of 96-well plate) in 10% FBS-RPMI 1640 and leave overnight in a 5% CO₂, 37°C incubator.
11. Change to fresh media the next morning and perform treatments as required.

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