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).
Protocol detail
Coculture of Myeloid Dendritic Cells
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DCP-MM medium
complete RPMI-1640 medium
IL-3
CD3-PE/FITC/APC
APC-Cy7
CD8-APC/FITC
CD56-PE
CD123-PE/PE-Cy5/FITC
CD138-FITC/PE/APC
BDCA-2-FITC
CD11c-APC
PD-L1-BV421
CellTrace Violet
CellTracker Green flow assay kits
Assay
Blocking antibody
Cd123
Cd138
Cell proliferation
Celltracker green
Clone
Enoblock
Fitc
Human
Immunomagnetic separation
Pd l1
Pdcs
Violet
Corresponding Organization :
Other organizations : Dana-Farber Cancer Institute
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Variable analysis
independent variables
- Culturing condition (DCP-MM medium or complete RPMI-1640 medium supplemented with IL-3)
- Blocking anti-human PD-L1 antibody (clone MIH1)
- HDAC6 inhibitor ACY241
- ENO inhibitor (ENOblock)
- Expression of cell surface markers (CD3, CD4, CD8, CD56, CD123, CD138, BDCA-2, CD11c, CD303, CD304, CD107a, PD-L1)
- Cell proliferation (measured by WST-1 Cell Proliferation Reagent)
- MM-pDCs (plasmacytoid dendritic cells from multiple myeloma patients)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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