Anti cd86 2331

Prior to staining, cells were washed in FACS buffer [PBS, 0.05% sodium azide with 0.5% bovine serum albumin (Sigma-Aldrich)]. Both during and after the staining procedure, cells were kept cold and in the dark. To be able to discriminate between live and dead cells in the subsequent analysis, cells were incubated with LIVE/DEAD Fixable Near-IR Stain (Invitrogen) according to manufacturer's instructions. DCs were additionally incubated with human IgG immunoglobulin (20 μg/mL, Kiovig Baxter) to block Fc receptors. The following fluorochrome labeled monoclonal antibodies were used for staining: anti-CD80 (L307.4, BD Pharmingen) and anti-CD86 (2331, BD Pharmingen). Cells were fixed in 1% paraformaldehyde and acquired within 5 days on a BD FACSCanto II (BD Biosciences). Unstained DCs were included in the analysis and used for calculating the mean fluorescence intensity (MFI) ratios between stained and unstained samples. Single-stained BD ComBead Plus compensation particles (BD Biosciences) were used for compensation. Data were analyzed using BD FACSDiva software version 8.0.2 (BD Biosciences).

A phenotypic analysis of human moDCs was performed for each donor after the differentiation of monocytes. Cells were stained using Zombie NIR viability dye, anti-CD14 (M5E2, BD Biosciences, Franklin Lakes, NJ, USA, RRID:AB_2687593), anti-CD209 (DCN46, BD Biosciences, Franklin Lakes, NJ, USA, RRID:AB_394123), and anti-HLA-DR (L243, BD Biosciences, Franklin Lakes, NJ, USA, RRID:AB_2738559). Cells were acquired using LSR II cytometers (BD Biosciences, Franklin Lakes, NJ, USA), and results were analyzed using FlowJo softwares (v10, Treestar, Woodburn, OR, USA). The activation state of moDC after their infection with rAAV was analyzed by flow cytometry using the following antibodies: anti-CD80 (L307.4, BD Biosciences, RRID:AB_10562564), anti CD86 (2331, BD Biosciences, Franklin Lakes, NJ, USA, RRID:AB_11153866).