Bone marrow derived macrophages (BMMΦs) were isolated from 6- to 8-week-old C57BL/6 mice as previously described [1 (link)] and cultured with 10 ng/mL GM-CSF (R&D) for 7 days to drive M1 macrophage maturation. The mouse macrophage cell line, RAW264.7, was also treated with GM-CSF (10 ng/mL) for 2 days to induce M1-like macrophages. All cells were maintained at 37°C in a 5% (v/v) CO2 enriched atmosphere in RPMI 1640 medium, supplemented with 10% (v/v) fetal bovine serum (FBS) containing penicillin and streptomycin.
To investigate TNFα production by mouse macrophages, mature M1 BMMΦs or RAW264.7 cells were first seeded at levels of 2 × 105 cells in the wells of a 24-well tissue culture plate. To these cells, IL-34 (R&D Systems) was added at 0, 10, or 100 ng/mL, and cells were then incubated with the added presence of 10 ng/mL GM-CSF for 5 (mature M1 BMMΦs) or 2 (RAW264.7 cells) days. After incubation, 1 × 105 HKC, which had been prepared as previously described [1 (link), 13 (link)], was added. Wells devoid of HKC served as negative controls. Cell culture supernatants were collected 24 h after stimulation and TNFα was detected using an enzyme-linked immunosorbent assay (ELISA).
To investigate TNFα production by mouse macrophages, mature M1 BMMΦs or RAW264.7 cells were first seeded at levels of 2 × 105 cells in the wells of a 24-well tissue culture plate. To these cells, IL-34 (R&D Systems) was added at 0, 10, or 100 ng/mL, and cells were then incubated with the added presence of 10 ng/mL GM-CSF for 5 (mature M1 BMMΦs) or 2 (RAW264.7 cells) days. After incubation, 1 × 105 HKC, which had been prepared as previously described [1 (link), 13 (link)], was added. Wells devoid of HKC served as negative controls. Cell culture supernatants were collected 24 h after stimulation and TNFα was detected using an enzyme-linked immunosorbent assay (ELISA).
Free full text: Click here
