Anti cd40 pe

Adherent cells after 5-day culture were collected after treatment with 5 mM EDTA-phosphate-buffered saline (PBS) containing 20% FCS. Cells were stained with anti-HLA-DR-PE, anti-CD80-PE, anti-CD40-PE, anti-CD36-APC or anti-E-cadherin-PE antibodies (BD Pharmingen) and acquired on a FACS Canto flow cytometer (Becton Dickinson).

Mature DCs were electroporated without or with IKKβ-RNA as described above and harvested 24 h, 48 h, or 72 h after transfection. Cells were stained at 4°C in FACS buffer for 30 min with the following antibodies: anti-CD40-FITC (BD), anti-CD40-PE (BD), anti-CD25-FITC (Cymbus Technologies, Southampton, Hampshire, United Kingdom or BD), anti-CD25-PE (BD), anti-CD70-PE (BD), anti-OX40L-PE (BD), anti-CD80-FITC (BD), anti-CD83-PE (Miltenyi), anti-CCR7-FITC (R&D Systems), anti-CD86-FITC (Cymbus Technologies), anti-CD86-PE (Miltenyi, BD), and anti-PD-L1-PE (eBioscience) and with matched isotype controls: IgG1-FITC (BD, Miltenyi), IgG1-PE (Miltenyi), IgG2a-FITC (BD), IgG3-PE (eBioscience). The cells were then washed once with FACS buffer and were taken up in FACS buffer or a mixture of equal amounts of FACS-Fix (DPBS with 2% formaldehyde) and FACS buffer. Afterwards, the immunofluorescence was determined using a FACScan cytofluorometer equipped with Cell Quest software (BD). The DCs were discriminated based on their size and granularity by gating in the forward and side scatter channels. The specific mean fluorescence intensities (specific MFI) were calculated by subtraction of the background mean fluorescence intensity obtained with the isotype control antibodies. All values were set in relation to the 24 h control condition to calculate the fold induction.

The An@AuNPs at a dose of 10 μg/mL was incubated with 5 × 10⁵ macrophages/well or BMDCs/well for 12 h. The macrophages or BMDCs stained with anti-CD86-APC-R700 (BD, cat. no. 565479), anti-CD80-FITC (BD, cat. No. 561954), and anti-CD40-PE (BD, cat. no. 553791) antibody were analyzed via flow cytometric examination.