Its medium supplement

Fatty acids (FAs) were purchased from Cayman and diluted in ethanol for all FAs [Palmitic acid (PA, C16:0), stearic acid(C18:0), palmitoleic acid (C16:1), oleic acid (OA, C18:1n-9), linoleic acid (LA, C18:2n6c), cis-8,11,14-eicosatrienoic acid (C20:3n6), cis-11,14,17- eicosatrienoic acid (C20:3n3), erucic acid (C22:1n9), eicosapentaenoic acid (EPA, C20:5n3), nervonic acid (C24:1n9), docosahexenoic acid (DHA, C22:6n3) and arachidonic acid (AA, C20:4n-6)] . The FAs-free medium was contained RPMI-1640 basic medium, 1×ITS medium supplement (Sigma) and 20 ng/ml GM-CSF without FAs supplement. Before addition to the culture medium, all FAs were conjugated to bovine serum albumin (BSA, fatty acid-free, Sigma) at the indicated concentration with FAs-free medium containing 1% BSA. The fatty acids were incubated with 1% BSA for 2 h before adding to DC. The concentration of saturated, monounsaturated, and polyunsaturated fatty acids in ITS medium was 20 µM. Half of the medium was discarded and replaced with fresh DC medium every other day. On day 6, DC were infected with the virus for 24 h and then analyzed by FACS.

Three-dimensional organotypic culture was performed in growth factor-reduced BM from Engelbreth–Holm–Swarm mouse sarcoma (Matrigel, Becton Dickinson), which was prepared according to the manufacturer's instructions. Isolated primary MMECs grown on low adhesion plates were trypsinized with 0.05% Trypsin-EDTA for 5-10 min, centrifuged and suspended with liquid Matrigel, and plated onto 8-well chamber slides at ∼1500 cells/well. Organoids were grown in DMEM/F12 supplemented with ITS medium supplement (Sigma-Aldrich), penicillin and streptomycin and 2.5 nM FGF-2 (Sigma-Aldrich). Wnt3a and Wnt4 (both R&D Biosystems) were used in 100 ng/ml, Rspo1 (R&D Biosystems) in 500 ng/ml, and porcupine inhibitor IWP-2 (Sigma-Aldrich) in 2 µM, and were added on the starting day of the cultures. Medium was refreshed every 3-4 days. For lactogenic differentiation, organoids were first grown for 6 days in the DMEM/F12 medium supplemented with 2.5 nM FGF-2, ITS and penicillin and streptomycin, and thereafter, 6 days in the same culture medium additionally supplemented with 1 μg/ml mouse prolactin (Sigma-Aldrich) and 1 μg/ml hydrocortisone (Sigma-Aldrich).