Gmscf

Approximately 50 mL of whole blood was diluted in phosphate buffered saline with 2 mM EDTA into 300 ml. The diluted blood was layered over Ficoll-Paque PLUS (GE Healthcare. Illinois, USA) and centrifuged to isolate peripheral blood mononuclear cells (PBMC). After lysing away any remnants of erythrocytes and removing platelets, CD14 microbeads were used to isolate monocytes through positive selection by magnetic cell sorting (Miltenyi Biotech. Bergisch Gladbach, Germany). The isolated monocytes were plated in a 24-well plate, ~0.7–1 × 10⁶ cells per well in 600 μL of RPMI with 100 U/mL penicillin, 100 μg/mL streptomycin, with 10% fetal bovine serum in each wells. Final concentrations of 500 U/mL of IL-4 (Peprotech. New Jersey, US) and 1000 U/mL of GM-SCF (Peprotech. New Jersey, US) were added to the media for 2 h after initial plating and at 3 days when media was changed. At 7 days, cells was changed into media without IL-4 and GM-CSF and continue culturing  2 h before stimulation. E. coli (strain O111:B4) LPS was added to final concentration of 0.1 μg/mL. The stimulated cells were harvested at 0, 1, 6, 12, and 24-h time points. mRNA was extracted using RNeasy Mini Kit (QIAGEN. Hilden, Germany). IL-1β expression levels (Hs01555410_m1) were measured and compared between WT homozyogote and V1 homozygote groups. GAPDH (Hs03929097_g1) was used as internal control for ∆∆Ct calculation.

For BMDC generation, bone marrow cells obtained from femurs and tibia of adult mice were cultured at 37°C and 5% CO₂ in RPMI 1640 (Hyclone) containing 10% FBS, 10mM HEPES, 2mM L-glutamine, 50mM 2-mercaptoethanol (Sigma), 50 U/ml penicillin, and 50ug/ml streptomycin. The culture medium was added with cytokines IL4 and GMSCF (PeproTech) on day 2. Day 5 BMDCs were harvest on day seven and exposed to 4mM tamibarotene, 500ng/ml IGF-1, 5ug/ml OVA, respectively, for experiments. For BMDC-T cell co-culture, naive T cells were isolated by CD4(+) negative separation Kit (Stemcells) from OT-II mice and co-cultured with BMDCs at a ratio of 1:2 in the presence of ova for three days. Cells were prepared for flow cytometry analysis.