Trypan blue

Required number of frozen vials of each donor was processed proportionally to the number of vials according to the following protocol. One vial containing 1 ml of cell suspension was placed into 37 °C water bath for 1 min. The cell suspension was transferred into 1 ml of prewarmed (37 °C) thawing medium prepared as 50% FCS, 50% HEK medium (90% RPMI medium, 10% FCS, 1% l-glutamine (200 mM) (Biochrom, Germany), 1% penicillin/streptomycin (10,000 U/ml) (Merck, Germany)) and 180 U/ml of DNAse I (Roche, Switzerland). Followed by immediate addition of 1 ml prewarmed (37 °C) HEK media. Cell suspension was placed for 1 hour in and 37 °C incubator. Afterwards, the cell suspension was centrifuged (360 × g, 6 min, RT) and the supernatant was discarded. Cells were resuspended in 1 ml HEK media and counted using trypan blue (ApplichemPanreac, Germany) and Neubauer chamber.

B cell subpopulations were sorted on a FACSAria™ Fusion (BD Biosciences, Heidelberg, Germany) using the version 8.0.1 of the BD FACS Diva software. Purity of sorted subpopulations was confirmed by remeasuring of samples. Sorted cells were washed, counted, and checked for viability using trypan blue (Applichem Panreac, Darmstadt, Germany). TNFR2-positive and -negative B cells were sorted from total B cells stimulated for 2 days by 1 µM CpG ODN 2006, if not stated otherwise.