Anti cd86 apc clone gl1

The biological activity of the NP-encapsulated agents was evaluated by seeding 5 × 10⁴ D1DCs in 96-well plates (Corning, Glendale, CA, USA) and incubated with the NPs for 48 h in an incubator. The NP concentrations were matched to poly(I:C) at 5 µg/mL and serially diluted, according to annotated concentrations, to establish a dose-response curve and enable comparison with the free ligand at 5 µg/mL. The cells were stained for the DC maturation markers CD86 and CD40, using anti-CD86-APC (clone GL1; eBioscience, Waltham, MA, USA) and anti-CD40-PE (clone 1C10; eBioscience, Waltham, MA, USA), respectively, and expression was measured by flow cytometry. The supernatant was collected, after which IL12 was analyzed by a standard sandwich ELISA, using the purified anti-mouse IL12/IL23 p40 (clone C15.6; Biolegend, San Diego, CA, USA) and biotin-labelled anti-mouse IL12/IL23p40 antibodies (clone C17.8; Biolegend, San Diego, CA, USA). The plates were read at 450 nm, using a Bio-Rad 680 microplate reader (Bio-Rad Laboratories, Veenendaal, The Netherlands).

Live cells were discriminated by a live/dead fixable aqua dead cell stain (Molecular Probes). For staining DCs, murine anti-CD11c PE-Cy7 (clone N418, eBioscience), anti-CD11b APC-Cy7 (clone M1/70, Biolegend), anti-CD40 PE-Cy5 (clone 1C10, eBioscience), anti-CD86 APC (clone GL1, eBioscience), and anti–MHC II PE (clone M5/114.15.2, BD) were utilized. For staining T cells, murine anti-CD3 V450 (clone 500A2, BD), anti-CD4 Alexa700 (clone RM4-5, BD), anti-CD8 PerCP (clone 53–6.7, BD), anti-TCR γδ BV605 (clone GL3, Biolegend), anti-CD44 APC-Cy7 (clone IM7, BD), anti-CD45.1 BV785 (clone A20, BioLegend), and anti-CD45.2 BV650 (clone 104, BioLegend) were utilized to stain for surface markers. Murine anti-RORγt PE (clone B2D, eBioscience), anti-TNFa PE-Cy7 (clone MP6-XT22, BD), anti-IFN-γ APC (clone XMG1.2, eBioscience), anti-IL-2 FITC (clone JES6-5H4, BD), and anti-IL-17 PE-CF594 (clone TC11-18H10, BD) were stained intracellularly with the BD Cytofix/Cytoperm or BD Transcription Factor kit as per manufacturer’s instructions. Staining for cell-surface markers was done by resuspending ∼1-2x10⁶ cells in 100 ml PBS with 2% FBS containing the antibody mixture at 4°C for 30 min and then washing with PBS containing 2% FBS. Data were immediately acquired using an LSRII flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (FlowJo LLC, Ashland, OR).