Cd11c bv421

DCs were loaded with OR141-killed cancer cells which were pre-labeled with CellTracker™ Green CMFDA Dye (Thermo Fisher Scientific Corp.). After 18 h incubation, DCs were collected and stained with CD11c-BV421 (BD Pharm, 565452) and propidium iodide (PI). Any instances of double positives for CD11c and CMFDA were analyzed on FACS Canto II.

The heparinized blood from each animal was divided into two parts. A volume of 150 µl was designated for blood count and measured using an ABX Pentra 60 C+ hemoanalyzer (Horiba, Kyoto, Japan). The rest of the blood (200–300 µl) was lysed using EasyLyse™ (Dako, Glostrup, Denmark). The amount of cells in suspension was as determined by Turk’s solution (2% acetic acid; Sigma-Aldrich) using a hemocytometer chamber. Cells (5 × 10⁵/100 µl) were marked with two panels of monoclonal antibodies. The first panel was delineated to detect T, B, and NK lymphocytes (CD3ϵ, CD4, CD8, CD19, and NK1.1). The second panel was designed for determination of monocytes and neutrophils (CD11b, F4/80, Ly6C, and Ly6G). The following monoclonal antibodies with fluorochromes for flow cytometry analysis were purchased from BioLegend (San Diego, CA, United States): anti-mouse CD3ϵ–FITC, CD4–BV421, CD19–PE, CD11c–BV421, CD45–APC/Fire™750, F4/80–PE, and from BD Bioscience: CD8–PECy7, CD11b–BV510, Ly6C–FITC, Ly6G–PECy7, and NK1.1–APC.

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)).

Leukocytes from lymph nodes, blood, spleen and peritoneal cavity wash were collected and isolated as described [56 (link)], and analysed by flow cytometry. Anti-mouse CD16/CD32 antibody (1:50, BD Biosciences, clone #2.4G2) was used to block non-specific binding. The following fluorochrome-conjugated antibodies were used: CD3e-AF700 (1:200, BD Biosciences, clone #500A2), CD4-BUV395 (1:100, BD Biosciences, clone #RM4-5), CD8a-PerCP (1:100, BioLegend, clone #53-6.7), CD25-PE-CF594 (1:100, BD Biosciences, #PC-61), FoxP3-APC (1:50, eBioscience, clone #FJK-16S), CD11b-APC (1:200, BioLegend, clone #MI/71), CD11c-BV421 (1:50, BD Bioscience, clone #HL3), GR1-PerCP Cy5.5 (1:200, BD Biosciences, clone #RB6-8C5), F4/80-BV711 (1:100, BioLegend, clone #BM8) and MHCII-APC/Cy7 (1:500, BioLegend, clone #M5/114.15.2). Single-stained beads were used to set compensation controls, and fluorescence-minus-one (FMO) controls were used to define population gates. Data were acquired using a BD LSRFortessa X-20 (BD Biosciences), and were analysed using FlowJo software v10.5.0 (BD Biosciences).