Generation and Characterization of Bone Marrow-Derived Dendritic Cells

Bone marrow-derived dendritic cells (BMDCs) were generated from Balb/CByJ or C57BL/6j mice as described previously [40 (link),61 (link)]. At day 7 post-isolation, BMDCs were either left untreated or exposed to CM treatment (dilution 1:1) for the indicated periods of time. In some experiments, DCs were exposed to 5 µM TGF-beta type I receptor inhibitor SB-431542, 15 µM DGAT1 inhibitor A922500, or 10 µM DGAT2 inhibitor PF-06424439; concentrations were chosen based on our previous work in cancer cells [36 (link)]. DC maturation was obtained by stimulation with 0.3 µg/mL LPS for 18 h and analyzed with antibodies against CD11c-BV421 (BD Pharm, 565452, San Jose, CA, USA), MHCII (I-A/I-E)-APC (BD Pharm, 565367), CD40-PE (BD Pharm, 553791), CD80-PE (eBioscience, 12-0801, San Diego, CA, USA), CD86-PE (eBioscience, 12-0862), or CCR7-PE (BioLegend 120105). Live/dead exclusion was achieved by staining with FVD eFluor780 (eBioscience, 65-0865-14). Flow cytometry analysis was performed on FACS Canto II and data were analyzed using FlowJo software (version 10.6.2). For DC vaccine generation, Ab1 cells were lysed by triple freeze–thaw processes and added to DCs at day 7. Tumor lysate-loaded DCs were treated on day 8 with 0.3 µg/mL LPS for 8 h before mouse i.p. administration (2 × 10⁶ DCs in 100 μL phosphate-buffered saline (PBS)).