For flow cytometry analysis of surface DC phenotype, we used the following monoclonal antibodies (mAb): FITC-labeled anti-CD1c (Biolegend, San Diego, CA, USA), anti-CD14, anti-CD16 and anti-HLA I (all from Invitrogen, Camarillo, CA, USA), Alexa Fluor 488-labeled anti-CD1a and anti-CCR7 (both Biolegend). PE-labeled antibodies included: anti-CD11b, anti-CD11c (both Biolegend), anti-CD80, anti-CD86, anti-DC-SIGN, (all Biolegend), and anti-HLA DR (Miltenyi Biotec, Bergisch Gladbach, Germany).
DCs differentiated in Cellgenix® DC GMP medium (Cellgenix GmbH, Freiburg, Germany) were harvested and collected by centrifugation. Before staining, the cells were washed twice in DPBS in all cases. Antibody was added and the cells were incubated for 15 min in the dark, then washed twice and resuspended in 2% paraformaldehyde (PHA). Samples were analyzed on a FACSCalibur system (BD biosciences, Franklin Lakes, NJ, USA). Data were analyzed with CellQuest software (BD biosciences).
DCs differentiated in Cellgenix® DC GMP medium (Cellgenix GmbH, Freiburg, Germany) were harvested and collected by centrifugation. Before staining, the cells were washed twice in DPBS in all cases. Antibody was added and the cells were incubated for 15 min in the dark, then washed twice and resuspended in 2% paraformaldehyde (PHA). Samples were analyzed on a FACSCalibur system (BD biosciences, Franklin Lakes, NJ, USA). Data were analyzed with CellQuest software (BD biosciences).