Blood processing and staining — immune monitoring panels and absolute count determination.
LYSING, LYSIS
All PB samples were subjected to high-throughput EuroFlow-based flow cytometric immunophenotyping with 4 multicolor immune monitoring panels (or their direct prototypes). These panels allow monitoring the kinetics of over 250 circulating immune cell subsets. In short, the B cell and plasma cell tube (BIGH) allows identification of up to 115 B and plasma cell subsets that are distinguished based on expressed Ig subclasses and their maturation stage–associated phenotype (All blood samples were processed according to the EuroFlow standard operating protocol (SOP) as reported previously (protocols available at In short, for CD4T and CYTOX panels, 100 μL PB was used for membrane and intracellular staining using the Fix and Perm Kit (Sanbio). For the BIGH and DC-Monocyte panel, high cell numbers were required, and thus a bulk lysis was performed on whole blood, and then 10 million cells were stained for membrane markers. For the BIGH panel, this was followed by intracellular staining for Igs (again, using the Fix and Perm Kit). For the DC-Monocyte panel, the surface staining was followed by a 10-minute incubation with FACS lysing solution (BD Biosciences), washed, and acquired on the flow cytometer.Absolute cell counts were determined with the use of Perfect-Count Microspheres (Cytognos) according to the EuroFlow SOP (All samples were measured on an LSR Fortessa 4L or on an LSR Fortessa X-20 4L (BD Biosciences). Flow cytometer performance was assessed daily according to the EuroFlow guidelines, as previously described (