Apc cy7 anti cd86

15dPMJ₂ and neutral-15dPMJ₂ were synthesized as described previously [13 (link)]. GSK2606414 was purchased from Millipore Sigma (Burlington, MA, USA). The reagents, 2-mercapthoethanol, propidium iodide and tetradecanoylphorbol acetate (TPA), were purchased from Sigma-Aldrich (St. Louis, MO, USA). GAPDH antibody was obtained from Cell Signaling Technologies (Beverley, MA, USA). Anti-CHOP10 was acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Caspase-Glo^® 3/7 assay kit, CellTiter-Glo 2.0 assay kit, and MTS reagent were purchased from Promega Life Sciences (Madison, WI, USA). Anti-calreticulin (Alexa Fluor 647) was from Abcam (Cambridge, MA, USA). HMGB1 ELISA kit was purchased from Novatein Biosciences (Woburn, MA, USA). Recombinant mouse GM-CSF, 7-AAD, PE-Cy7-anti-CD80, APC-Cy7-anti-CD86, PE-Anti-Hsp90 and FITC-Anti-Hsp70 were obtained from BioLegend (San Diego, CA, USA). APC-anti-CD11c and mouse Fc block were purchased from BD Biosciences (San Jose, CA, USA). FITC-anti-MHC class II, anti-CRT and CellTracker Green CMFDA were purchased from Thermo Fisher Scientific (Waltham, MA, USA). IRDye 800CW anti-rabbit and IRDye 680RD anti-mouse secondary antibodies were from LI-COR Biosciences (Lincoln, NE, USA).

BMDCs or splenic cells were incubated for 15 min at room temperature with 5 μg/mL anti-CD16/32 antibody (Fc Block; clone: 93) (BioLegend), to avoid non-specific staining, and 7-AAD Viability Staining Solution (BioLegend), to detect dead cells. BMDCs or splenic cells were then stained for 30 min at 4 °C with following antibodies. For BMDCs, FITC-anti-I-A^d (BioLegend; clone: 39-10-8), PE-anti-CD80 (BioLegend; clone: 16-10A1), APC-Cy7-anti-CD86 (BioLegend; clone: GL-1), and PE-Cy7-anti-CD40 (BioLegend; clone: 3/23) were used. For splenic cells, FITC-anti-I-A^d (BioLegend; clone: 39-10-8), PE-anti-CD80 (BioLegend; clone: 16-10A1), and Brilliant Violet 421-anti-CD11c (BioLegend; clone: N418) were used. Samples were analyzed with a MACSQuant^® Analyzer (Miltenyi Biotec). Flow cytometry data were analyzed with FlowJo, LLC Software 10.2 (BD Biosciences).