Macrophage colony stimulating factor m csf

Femora and tibiae were dissected and cleaned mechanically using paper tissue to remove the surrounding muscle tissue. Bones were placed in cold PBS in a petri dish and transferred to a sterile hood. After soaking for 30 s in 70% ethanol and two washes in sterile PBS, the contents of bone marrow cavities were flushed out with cold PBS using a syringe and a needle. Eluates were passed through a 70 µm cell strainer into falcon tubes and centrifuged at 300 g for 8 min at 4°C. Supernatant was discarded and each cell pellet was re-suspended in 3 ml ACK lysis buffer (Life technologies Ltd.) and incubated for 5–10 min at room temperature (RT) for red blood cell elimination. 3ml PBS was added to stop the reaction and cells were centrifuged at 300 g for 6 min. Supernatant was discarded and each pellet re-suspended in 10 ml DMEM F12 with 10% (v/v) FBS, 2 mM L- Glutamine, 100U/ml Penicillin, 100 µg/ml Streptomycin and 20 ng/ml Macrophage-colony stimulating factor (M-CSF) (Sigma Aldrich Ltd.). Suspensions were plated in 10 mm polystyrene petri dishes or 6-well tissue culture plates (Fisher Scientific Ltd.) and incubated at 37°C in a humidified environment of 5% CO₂. After 3 days in culture, 3 ml of fresh media was added to each petri dish or well. On day 7, non-adherent cells were removed, and adherent cells were considered macrophages.

The compounds of Kushennol F (KF) and Sohoravanone G (SG) were isolated and identified as previously described [21 (link)]. Cell culture medium including Alpha-modified Minimum Essential Medium Eagle (α-MEM), Phenol Red Free α-MEM, Dulbecco’s Modified Eagle’s Medium (DMEM), and cell culture serum including Fetal Bovine Serum (FBS) and Dextran-charcoal-stripped Fetal Bovine Serum (sFBS) were purchased from Gibco (Gibco, Gaithersburg, MD, USA). Penicillin and streptomycin were purchased from Invitrogen (Invitrogen, Carlsbad, CA, USA). 17β-estradiol (E₂), receptor activator of nuclear factor κB (NF-κB) ligand (sRANKL), and Macrophage Colony-Stimulating Factor (M-CSF) were purchased from Sigma Aldrich (Sigma, St. Louis, MO, USA).