Igg3 pe

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.