Csf 1

Hippocampus samples were extracted and homogenized in lysis buffer containing protease and phosphatase inhibitors, and the homogenates were centrifuged at 7000 g for 15 mins at 4°C as described previously (Wu et al. 2012 (link)). The supernatants were collected and the protein concentration was determined by Bradford method, and 30 μg of proteins were loaded for each lane and separated by SDS-PAGE. After the transfer, the blots were incubated overnight at 4°C with one of the primary antibodies listed below: CSF-1 (1:500, R&D, AF416), β-actin (1:5000, rabbit; Santa Cruz). These blots were then washed, incubated with HRP-conjugated secondary antibody and developed in an enhanced chemiluminescent solution. Specific bands were evaluated by apparent molecular size. Finally, images were captured using a Fuji-Film LAS-300 (Fuji, Sheffield, UK) and the intensity of the selected bands was analyzed using Image J software.

Highly metastatic MDA-MB-231 breast adenocarcinoma cells or MET-1 cells extracted from MMTV-PyMT female mice were maintained in DMEM supplemented with 10% FBS (Atlanta Biologicals, Flowery Branch, GA, USA) and 1% penicillin-streptomycin. BAC1.2F5 cells (generously provided by Dr. Richard Stanley, Albert Einstein College of Medicine) were cultured in α-MEM supplemented with 10% FBS, 1% penicillin-streptomycin, and 3000 U/mL of CSF-1 (R&D System, Minneapolis, MN, USA). To produce lentivirus particles, HEK293T cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. All cells were cultured at 37°C and 5% CO₂. All cell lines were tested and found negative for mycoplasma contamination using a universal mycoplasma detection kit (30–1012K, ATCC). For FAK inhibition studies, cells were treated with 10 μM PF573228.

Bone marrow cells were isolated from tibias and femurs of WT mice and grown in complete medium (RPMI-1640 medium containing 10 mM glucose, 2 mM L-glutamine, 100 U ml^-1 penicillin-streptomycin and 10% FCS) with 20 ng ml^-1 murine macrophage colonystimulating factor 1 (CSF-1; Peprotech) for 7 days, and supplemented with CSF-1 on days 3 and 5. On day 7 macrophages were harvested and then maintained in 20 ng ml^-1 CSF-1 for subsequent experiments in which they were either maintained in medium alone (M0), or stimulated with 50 ng ml^-1 IFN-μ (R&D systems) and 20 ng ml^-1 LPS (M1), or 20 ng ml^-1 IL-4 (M2) (Peprotech) for 18 h.

Frozen PBMC isolated from healthy donors were thawed briefly at 37°C in a water bath, washed in R-PS [RPMI1640 containing 50 penicillin (µg/mL) and streptomycin (100 µg/mL)], and incubated in R-PS containing 5% FBS (R-PS5) on cell culture dishes for 1.5 h. Floating cells were removed by washing with R-PS at least 5 times. The attached monocytes were cultured in R-PS10 medium (R-PS supplemented with 10% FBS) containing CSF1 (15–100 ng/ml; Biolegend) for 3 d to differentiate into macrophages. After being washed with R-PS, the attached monocyte-derived macrophages (MDM) were incubated with fresh R-PS10 containing CSF1 for 3 more days. MDM were activated with CSF1 (100 ng/mL), IL4 (10 or 20 ng/mL, R&D Systems), or IL10 (10 or 20 ng/ml, R&D Systems) for 2 days. In some experiments, ANGPT2 (300 ng/mL; R&D Systems and EMD Millipore) was added to MDM after 3 d of differentiation with CSF1 or when they were activated with IL4 or IL10 to examine its effect on PD-L1 expression. Phenotypes of polarized MDM were analyzed by FACS after staining with APC-conjugated CD80 (Clone 2D-10, Biolegend) and PE-conjugated CD163 antibodies (Clone GHI/61, Biolegend).

Peripheral blood mononuclear cells were isolated from buffy coats of healthy donors with a Ficoll (GE) gradient using SepMate tubes (StemCell) and monocytes selected by MACsorting with CD14 MicroBeads (Miltenyi). Monocytes were differentiated into macrophages by culture in Teflon-coated bags (OriGen) for 7 days in DMEM +GlutaMAX (Gibco) +10% FBS +1% P/S with the addition of 10 ng/ml recombinant human CSF-1 (R&D Systems).
Differentiated MDMs were plated at a density of 1 × 10⁶ cells/well of a 6-well plate in DMEM +10% FBS +1% P/S +10ng/ml CSF-1. After cell attachment, MDMs were cultured in serum free medium for 6 hours before stimulation with TME-CM for 24 hours.