Crpmi 1640

α,α′-Trehalose 6,6′-glycolipids, AF-2 and TDB, were made up to 1 mM in CHCl₃/MeOH (2/1, v/v), then diluted to 400 μM or 50 μM in isopropanol to give 8 or 1 nmol of glycolipid per 20 μL, respectively. The solutions were then added to 96-well plates (20 µL/well) and the solvent was evaporated within a sterile hood for 18 h.
Bone marrow cells were collected from the tibia and femur of C57BL/6 or Mincle^−/− mice and cultured (250,000 cells/mL) in complete Roswell Park Memorial Institute medium [cRPMI-1640 (Gibco) with 10% heat inactivated fetal bovine serum (Gibco), 100 unit/mL penicillin-streptomycin (Gibco) and 2 mM Glutamax (Gibco)]. Macrophage differentiation was induced by 50 ng/mL GM-CSF (PeproTech) added to the cRPMI. Cells were incubated at 37 °C (5% CO₂) for 8 days (cells fed on days 3 and 6). On day 8, the media was removed, the cells were lifted using Accutase (StemCell), counted and re-seeded in 96-well plates coated with ligands. Where indicated Ac-YVAD-cmk (40 μM, Sigma) or KCl (50 mM) [12 (link)] were added to the cells an hour before, or CY-09 (20 or 40 µM) was added 30 min before adding the cells to ligand-coated plate according to the manufacturer’s protocol. 100 ng/mL LPS (Sigma) or nigericin (10 μM, ChemImpex) were used as controls [29 (link)].

The human myeloid leukemia cell line (K562) was cultured in complete Roswell Park Memorial Institute Medium (cRPMI 1640) (Gibco). Packaging cells (293T/17) were cultured in complete Dulbecco's modified Eagle's medium (cDMEM) (Gibco). Culture media were supplemented with 10% fetal bovine serum (FBS) (Hyclone), penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were maintained in a humidified incubator at 37°C and 5% CO₂. cDMEM media without antibiotics were used for further lentiviral production.

As previously reported, PBMCs and human-derived macrophages (MDMs) were isolated or differentiated (37 (link), 38 (link)). Briefly, PBMCs were enriched from whole blood by density gradient using Histopaque-1077 (Sigma-Aldrich, UK). Cells were washed twice in PBS and resuspended in Complete Roswell Park Memorial Institute Medium (cRPMI-1640) culture medium (RPMI 1640 with 2 mM glutamine; Gibco, Thermo Fisher Scientific, USA), 10% human serum (Sigma-Aldrich, USA), 10 U/mL penicillin/streptomycin, and 10 mM HEPES (Thermo Fisher Scientific, the Netherlands). Monocytes (CD14⁺) were isolated from PBMCs by positive selection using magnetically labeled CD14 Microbeads (Miltenyi Biotec). The isolated monocytes were then resuspended in cRPMI medium with 20 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF, Miltenyi Biotec), and cells were seeded at a concentration of 1 × 10⁶ cells/mL in 24-well and 48-well plates (Corning Inc., South Korea) for 7 days. The culture medium was refreshed on the fourth day of incubation. Before infection, the acquisition of macrophage morphology was confirmed by visualization in a BX61 microscope (Olympus).