Gm csf

The concentration of BM cells was adjusted to 5 × 10⁵ cells/mL in CM. GM-CSF (PeproTech, Rocky Hill, NJ, USA) was added to the cell suspension at a final concentration of 10 ng/mL. Where stated, IL-4 (PeproTech, Rocky Hill, NJ, USA) was also added to the cultures at a final concentration of 5 ng/mL. BM cells were cultured in 24-well plates in 1 mL of CM with GM-CSF or GM-CSF + IL-4 and incubated at 37 °C in 5% CO₂. Cells were harvested by gentle resuspension on either day 3, 4 or 5 unless otherwise stated. To harvest the cells, the plates were centrifuged at 1400 rpm for 5 min at 4 °C. The supernatant was collected, and cells were re-suspended in phosphate buffered saline (PBS) and prepared for cell surface staining.

Monocytes were cultured in RPMI 1640 medium (Lonza) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific), 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine and 1 mM pyruvate (all from Lonza), referred to as complete-RPMI medium, in the presence of GM-CSF (5 ng/mL; Gibco, Thermo Fisher Scientific) to induce M1 macrophages or with GM-CSF (20 ng/mL) and IL- 4 (20 ng/mL; Gibco, Thermo Fisher Scientific) to induce DC differentiation. After 3 days, additional 5 ng/mL GM-CSF was added to macrophage cultures and half of the medium was renewed in DC cultures. At day 6 of co-culture, macrophage and DC phenotypes were analyzed by flow cytometry within CD90^- population.
Macrophages and DCs were stimulated overnight with LPS (Invitrogen, Life Technologies) at 10 ng/mL or 50 ng/mL, respectively. Supernatants were collected and their allostimulatory function was analyzed by culturing in mixed lymphocyte reaction (MLR) with CFSE-labeled T lymphocytes (1:10 Macrophage or DC/T cell ratio). After 5 days of co-culture, T lymphocyte proliferation was analyzed using the CFSE dilution method by flow cytometry in the CD3⁺ population. Supernatants from different co-cultures were harvested at different times and cytokine secretion was measured.

Human PBMC were separated from buffy coats of healthy donors (Hong Kong Red Cross Blood Transfusion Service) using Ficoll-Paque density gradient (GE Healthcare Life Sciences) under Institutional Review Board (IRB) approval (UW 10-124). Monocytes/macrophages were isolated by plastic adherence and were cultured in complete RPMI medium supplemented with 10% fetal bovine serum (FBS) and 10 ng/ml human M-CSF or GM-CSF (Gibco) for 7 days. Human PBMC-derived dendritic cells were differentiated with 10 ng/ml of IL-4 (Gibco) and 50 ng/ml of GM-CSF for 7 days. The research protocol was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW 10-124). For preparation of murine bone marrow-derived macrophages, bone marrow cells were isolated from femurs and tibias and cultured in RPMI medium supplemented with 10% FBS and 10 ng/ml of mouse M-CSF (R&D Systems) for 7 days.