Recombinant mouse m csf

Scaffolds containing p53^KOCcne1⁺ tumor cells were transplanted subcutaneously into in p53^KO mice to generate sarcoma tumors. Tumors were dissociated and immortalized p53^KO CAF were FACS sorted as follows: glyCAF (Live, dsRED⁻, CD31⁻, CD45⁻ CD90⁺ CD73⁺ cells) and non-glyCAF (Live, dsRED⁻, CD31⁻, CD45⁻ CD90⁺ CD73⁻ cells). Individual cells were isolated by serial dilution, expanded, and then validated for CAF markers by positive expression of CD90 and lack of tumor dsRED by flow cytometry. The sorted cells were maintained in DMEM supplemented with glutamine, 10% fetal bovine serum, 100IU/ml penicillin and 100 μg/ml streptomycin (Gibco) and maintained at 37 °C and 5% CO₂. Bone marrow-derived macrophages were generated by isolating total hematopoietic cells from mouse bone marrow and stimulated in vitro with recombinant mouse M-CSF (BioLegend) for 6 days. New M-CSF was added every other day with fresh medium.

Recombinant mouse M-CSF, GM-CSF, IL-4, and RANKL were purchased from BioLegend (San Diego, CA). AF488-phalloidin was purchased from Invitrogen (Carlsbad, CA). Antibodies for immunoblot assays were obtained against NFATc1 (Pierce, Rockford, IL); Oscar (R&D Systems, Minneapolis, MN); TRAP (BioLegend); Integrin β3; c-Fos; cleaved Caspase-3, -8, and -9; and cleaved PARP (Cell Signaling Technology, Mountain View, CA). The previously described anti-Ninj1 Ab_1-15^{18 (link)} was used for immunoblotting assays. For FACS analysis, Fc block, and PE-anti-mouse CD115, APC-anti-mouse CD117, and V450-anti-mouse CD11b antibodies were purchased from BD Biosciences (Bedford, MA), and biotin-anti-RANK antibody and APC/Cy7 streptavidin were obtained from BioLegend.

The antibodies used in this study are summarized in Supplemental Table S2. Recombinant mouse M-CSF (#576404), IL-10 (#575806) and IL-6 (#575702) were obtained from Biolegend. Recombinant mouse GM-CSF (#ab198564) was purchased from Abcam. Recombinant human IL-10 (#200-10) was obtained from PeproTech. Recombinant mouse IFN-γ (#21-8311) and IL-4 (#21-8041) were purchased from TONBO biosciences. Oil Red O (#O0625) and LPS (#L2630) were obtained from Sigma-Aldrich.

If not otherwise stated throughout the main text or in each figure caption, bone marrow (BM)-derived macrophages (BMDMs) were prepared from 7- to 9-week-old CO₂-euthanized C57BL/6N female mice and subjected to red blood cell lysis with ACK lysis buffer prior to neutralization in phosphate-buffered saline (PBS). BM was seeded at 3 × 10⁵ cells/well in six-well tissue-culture plates and differentiated in IMDM medium (Thermo Fisher Scientific) containing 25 mM glucose supplemented with 10% FBS (Gibco), 2 mM l-l-glutamine (Thermo Fisher Scientific) in the presence of 20 ng/ml recombinant mouse M-CSF (576404, Biolegend) for 7 days. When BMDMs from Balb/c background were used, they were generated analogously to C57BL/6N ones.

Bone marrow was collected from 12- to 16-week-old female wild-type C57BL/6 mice (experiments approved by the animal experiments committee of the Academic Medical Center, Amsterdam) and cultured in 100 mm cell culture dishes (BD Falcon) in IMDM (Lonza) supplemented with 10% FBS, 86 μg ml⁻¹ gentamicin and 40 ng ml⁻¹ recombinant mouse M-CSF (Biolegend). At day 3, fresh medium containing M-CSF was added. At day 6, M-CSF and either 10 ng ml⁻¹ recombinant mouse IL-10 or 20 ng ml⁻¹ recombinant mouse IL-4 (both from eBioscience) were added. At day 7, cells were harvested and stimulated as described for human macrophages, using 2 μg ml⁻¹ mouse IgG (Equitech-Bio) coated in Maxisorp plates. Cells were analysed by quantitative RT–PCR as described; primer pairs are listed in Supplementary Table 2.