Isolated PBMCs were stained with optimal dilutions of CD3-APC-H7 (Clone: SK7; BD Biosciences), γδTCR-APC (Clone: B1; BD Biosciences), CD38-PE (Clone: IB6; Miltenyi Biotec), Ki67-V450 (Clone: B56; BD Biosciences). Prior to staining with antibodies, unspecific antibody binding was blocked with Beriglobin (ZLB Behring) at a final concentration of 0.01 mg/ml. Surface staining was performed in PBS at 107 cells/ml for 15 minutes at room temperature. Stained cells were fixed with the paraformaldehyde containing FACS-Lysing-Solution (BD Biosciences) for 10 minutes at room temperature in the dark. After permeabilization with FACS-Perm-Solution (BD Biosciences) for 10 minutes at room temperature, Ki67 was stained intra-cellular for 30 minutes at room temperature in the dark in FACS-Perm-Solution.
The cells were acquired on a MACS Quant (Miltenyi Biotec) flow cytometer. Quality control was performed daily using SPHERO Rainbow Calibration Particles (BD Biosciences). No adjustment to the PMT voltage was required between the runs. A compensation matrix was established the day before vaccination using single antibody stains.
The cells were acquired on a MACS Quant (Miltenyi Biotec) flow cytometer. Quality control was performed daily using SPHERO Rainbow Calibration Particles (BD Biosciences). No adjustment to the PMT voltage was required between the runs. A compensation matrix was established the day before vaccination using single antibody stains.