Investigating PCP Uptake in Dendritic Cells

PCP was labeled with fluorescein isothiocyanate (FITC) via tyramine reduction method as previously reported [20 (link)]. For uptake assay, DC2.4 cells were seeded in 96-well plates and incubated PCP-FITC at 200, 400 and 800 μg/mL, BMDCs were cultured with PCP-FITC at 50, 100, 200 μg/mL. After 30 min, cells were harvested and stained with anti-mouse CD11c antibody before FACS analysis. For uptake competition assay, BMDCs were pre-incubated with 200 μg/mL Fucoidan (Solarbio, China), 20 μg/mL anti-mouse Dectin-1 antibody (InVivoGen, US), 200 μg/mL Laminarin (InVivoGen, US), 200 μg/mL D-Galactose (Aladdin Bio-Chem Technology, China) or 200 μg/mL Mannan (Solarbio, China). After 30 min, BMDCs were cultured with PCP-FITC (100 μg/mL) for 45 min before FACS analysis. For antigen uptake, BMDCs were first incubated with PCP (200 μg/mL) for 30 min, then DCs were co-cultured with OVA-atto 488 (5 μg/ml), Dextran-FITC (MW 40,000, 100 μg/ml) or Lucifer yellow VS dilithium salt (LY, 100 μg/ml) for 45 min before analysis of antigen uptake by CD11c⁺ BMDCs.