Anti cd11c apc

Phenotypical assessment of DC maturation after 48 h of co-culture with tumor cells was performed by flow cytometry. Briefly, cells were washed in PBA, incubated with Fc-receptor blocking buffer (2% HS in PBS, 15 min at 4°C) and subsequently stained with primary antibodies in PBA (30 min at 4°C). Monoclonal directly labeled anti-human antibodies used were (Table S1): anti-CD11c-APC (Miltenyi biotec, clone MJ4-27G12, catalog# 130-092-412), anti-CD123-APC (Miltenyi biotec, clone AC145, catalog# 130-090-901), anti-CD80-PECy7 (BD PharMingen, clone L307.4, catalog# 561135), anti-CD86-PECy7 (BD PharMingen, clone 2331, catalog# 561128), anti-HLA-ABC-PE (BD PharMingen, clone G46-2.6, catalog# 555553), and anti-HLA-DR-PE (BD PharMingen, clone G46-6, catalog# 555812). Appropriate isotype controls were included. Geometric mean fluorescence intensity (GeoMFI) of maturation markers was assessed on CD11c⁺ (for CD1c⁺ and CD16⁺ DCs) or CD123⁺ (for pDCs) populations. As a positive control, DCs were stimulated with poly I:C (CD1c⁺ and CD16⁺ DCs, 2 µg/mL, Enzo Life Science, catalog# ALX-746-021-M005) or R848 (pDCs, 4 µg/mL, Enzo Life Science, catalog# ALX-420-038-M025). To improve in vitro pDC viability, IL-3 (10 ng/mL, Cellgenix, catalog# 1002-050) was added to culture medium.

Both adherent and suspended cells cultured by the above methods were harvested from the 12-well plate and centrifuged at 1500 rpm for 5 min at 4 °C. After centrifugation, the cells were washed and resuspended using fluorescence-activated cell sorting (FACS) buffer. Afterwards, the cells were stained with the surface marker antibodies anti-CD11c-APC and anti-CD86-VioBlue (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 min at 4 °C. The cells were washed again with FACS buffer and analyzed by a FACS instrument (MACSQuant VYB; Miltenyi Biotec). CD11c and CD86 cell populations were measured, and activation levels of dendritic cells were calculated as CD86⁺ cells among CD11c⁺ cells within the living cell region.

BMDCs that were cultured in a 12-well plate using the above methods were used to measure the phagocytic activity of DCs. FITC-conjugated polystyrene beads (Sigma-Aldrich, St. Louis, MO), 1.80–2.20 μm in size, were incubated with both control cells and LPS-treated cells for 6 h at 37 °C. After the incubation, both adherent and suspended cells and PS beads were collected from the plate and centrifuged at 1500 rpm for 5 min at 4 °C. Then, the cells were washed with FACS buffer and stained with the cell surface marker antibodies anti-CD11c-APC and anti-CD86-Vioblue (Miltenyi Biotec) for 10 min at 4 °C. After staining, the cells and PS beads were washed again with FACS buffer and analyzed by a FACS instrument (MACSQuant VYB; Miltenyi Biotec). CD11c and CD86 cell populations and the PS beads internalized by CD11c and CD86 cell populations were measured. Activation levels and uptake levels of dendritic cells were calculated for the living cell region.