Anti bdca2

Blood samples were obtained before the surgery as well as 24 and 48 hours after the procedure. Mononuclear cells were separated on lymphocyte separation medium – Gradisol L (Aqua Med, Poland) and centrifuged for 20 minutes at 700 × g. Every 10⁷ of mononuclear cells were incubated with 10 µl of FcR – blocking reagent (Miltenyi Biotec, Germany) for 5 minutes to avoid non-specific binding and labeled with monoclonal antibodies against following antigens: BDCA-1, BDCA-2, and CD19. After incubation, cells were centrifuged and washed for 5 minutes at 700 × γ in 4 degrees of Celsius. The myeloid dendritic cells: anti – BDCA-1 (CD1c) FITC (Miltenyi Biotec, Germany) and anti – CD19 CyChrome (Becton Dickinson, USA). The lymphoid dendritic cells: anti – BDCA-2 (Miltenyi Biotec, Germany) and CD123 PE (Becton Dickinson, USA). The cells were collected using a FACSCalibur flow cytometry (Becton Dickinson, USA) and the phenotypes were analyzed with CellQuest software (Becton Dickinson, USA). For each analysis, 300,000 of events were collected. Cell debris and dead cells were excluded based on scatter signal. The fluorescence-minus-one method or the unstained cell were applied as negative control.

After blood collection, peripheral blood mononuclear cells (PBMC) were prepared by Ficoll-Hypaque (Biochrom, Berlin, Germany) density centrifugation. Subpopulations of T cells, B cells, natural killer (NK) cells, and antigen-presenting cells (APC) were characterized by surface staining with fluorescence-labeled anti-CD3, anti-CD4, anti-CD8, anti-CD16, anti-CD14, anti-CD19, anti-CD56 (BD Biosciences, Heidelberg, Germany) or anti-BDCA2, and anti-slan (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer's instructions. Negative controls included directly labeled or unlabeled isotype-matched irrelevant antibodies (BD Biosciences). Cells were evaluated on LSR-Fortessa (BD Biosciences).