Recombinant csf 1

Human FRCs at passage 3 were plated overnight at 2 × 10⁴ cells/0.32 cm² well. The following day, 4 × 10⁵ human PBMCs were isolated from healthy donors using Ficoll‐Paque or Lymphoprep according to the manufacturer's instructions and added to appropriate wells. Where indicated, 0.1 µg/mL human anti‐CSF1R (Clone 61701, R&D systems) or isotype control (Clone 11711, R&D systems) was added. Cells were incubated for 72 h at 37°C, then harvested, quantified, labeled with antibodies (Table 1), and analyzed by flow cytometry (Supporting information Fig. S3C). For mouse assays, 2 × 10⁵ mouse FRCs were left to adhere to six‐well plates overnight. The next day 1 × 10⁶ mouse BM cells were added to each well, with 100 U/mL recombinant CSF1 (Peprotech) or 10 µg/mL purified anti‐mouse CSF1R/CD115 blocking antibody (Clone AFS98, eBioscience). Cells were harvested after 4 days, quantified using a Z2 Coulter Counter (Beckman Coulter, USA), labelled for flow cytometry, and then analyzed on a FACS Canto (BD Biosciences) using Flowlogic software version 1.7 (Inivai Technologies) or Flowjo software v10 BD Biosciences (Supporting information Fig. S3A).

CSF-1 concentrations were analyzed by standard sandwich ELISA. The capture antibody (clone 5A1) was used at 1 ug/ml. Recombinant CSF-1 (Peprotech) was used as a standard. The detection antibody (biotinylated polyclonal anti-CSF-1, R and D systems cat # BAF416) was used at 0.5 ug/ml. Avidin-HRP and TMB substrate (ebioscience) were used for detection. To quantify CSF-1 levels in tumor microenvironments, tumors were dissociated as described above, and the supernatants from the dissociation were subjected to CSF-1 ELISA; intra-tumor concentrations were calculated according to measured tumor volumes calculated using the modified ellipsoid formula: V = 0.5 x [(length + width)/2]³, and the volume of dissociation supernatant.

Recombinant CSF-1, IL-10, and CCL5 were obtained from Peprotech (London, UK), GW2580 (anti- CSF-1) from Sigma-Aldrich (St. Louis, Missouri), anti-IL-10 from BDBiosciences, Lipopolisaccharide (LPS) from InvivoGen (tlrl-pelps, San Diego, CA, USA), and anti-CCL5 from Antibodies-online GmbH (Aachen, Germany). All concentrations were used such as it were described in previous works and tested previously in HD donors^{9 , 50 (link)–52 (link)}. Cells were stained with LIVE/DEAD fixable dead cell stain kit (Invitrogen, Carlsbad, CA, USA) to test viability after 24 h of culture with GW2580. Only samples with >95% of cellular viability were included.