Cd123 pe pe cy5 fitc

MM cells were cultured in 10% FBS plus RPMI-1640 medium supplemented with antibiotics. MM–pDCs were cocultured either in DCP-MM medium (Mattek Corp., Ashland, MA) or complete RPMI-1640 medium supplemented with IL-3 (Peprotech Inc., Rocky Hill, NJ, USA). CD3-PE/FITC/APC; CD4-FITC/PE or APC-Cy7; CD8-APC/FITC, CD56-PE; CD123-PE/PE-Cy5/FITC; and CD138-FITC/PE/APC were obtained from BD Biosciences (San Jose, CA). BDCA-2-FITC and CD11c-APC were obtained from Miltenyi Biotec (Auburn, CA); CD303-; CD304-; CD107a; and PD-L1-BV421 were purchased from Biolegend. All immunomagnetic separation kits were purchased from Miltenyi Biotec. The CellTrace Violet and CellTracker Green flow assay kits were obtained from Life Technologies (USA). Functional-grade PD-L1 blocking antibody (antihuman PD-L1, clone MIH1) was obtained from eBiosciences [4 (link)]. Ro 61–8048 [13 (link)] and INCB 024360 were purchased from Selleck Chemicals. WST-1 Cell Proliferation Reagent was purchased from Clontech Laboratories, Inc. (USA).

MM cell lines and PBMCs from normal healthy donors were cultured in RPMI-1640 medium supplemented with 10% FBS and antibiotics. Human recombinant IL-3 was purchased from Peprotech Inc (Rocky Hill, NJ, USA). CD3-PE; CD4-FITC or APC-Cy7; CD8-APC, CD123-PE/PE-Cy5/FITC; CD138-FITC/PE/APC; CD133-PE and CD27-Alexa-700 were obtained from BD Biosciences (San Jose, CA, USA). HLA-DR Violet Blue, CD303 (BDCA-2)-FITC, CD14-PE, and CD11c-APC were purchased from Miltenyi Biotec (Auburn, CA, USA); CD304-BV421 was obtained from Biolegend. All immunomagnetic separation kits were purchased from Miltenyi Biotec. Bortezomib, lenalidomide, SAHA, dexamethasone and pomalidomide were purchased from Selleck Chemicals, LLC (Houston, TX, USA).

MM-pDCs were cocultured either in DCP-MM medium (Mattek Corp. Ashland, MA) or complete RPMI-1640 medium supplemented with IL-3 (Peprotech Inc., Rocky Hill, NJ, USA). CD3-PE/FITC/APC; CD4-FITC/PE or APC-Cy7; CD8-APC/FITC, CD56-PE; CD123-PE/PE-Cy5/FITC; and CD138-FITC/PE/APC were obtained from BD Biosciences (San Jose, CA). BDCA-2-FITC and CD11c-APC were obtained from Miltenyi Biotec (Auburn, CA); CD303-, CD304-, CD107a, and PD-L1-BV421 were purchased from Biolegend. Immunomagnetic separation kits were purchased from Miltenyi Biotec. The CellTrace Violet and CellTracker Green flow assay kits were obtained from Life Technologies (USA). PD-L1 blocking antibody (anti-human PD-L1, clone MIH1) was obtained from eBiosciences [19 (link)]. HDAC6 inhibitor ACY241 and ENO inhibitor (ENOblock) [40 (link)] were purchased from Selleck Chemicals. WST-1 Cell Proliferation Reagent was purchased from Clontech Laboratories, Inc. (USA).