Cd27 pecf594

To sort antigen-specific cells, tetramers were generated as described in Franz et al. (52 (link)). A mix of Neutravidin (Neutravidin DyLight650, Thermo Scientific) and biotinylated peptide was incubated in the dark on ice for 30 min. Aggregates were removed by high speed centrifugation. B cells from acute Borreliosis patients were stained for 30 min with a pool of the three peptide tetramers and washed twice with FACS buffer (PBS, 2% FBS). The following antibodies were used to distinguish the different memory B cell subpopulations and to gate out monocytes, T-cells and dead cells: CD14-FITC, CD3-FITC (Immunotools), IgD-BV421, CD27-PECF594 (BD Horizon), CD19-BV605 (BDPharmingen), and live/dead marker. CD20-Biotin (Immunotools) was used as a compensation control for Neutravidin. Single cells were sorted on a FACSAria SORP machine (BD Biosciences) into 96-well PCR plates (Eppendorf) containing 5μl of 0.5% PBS, 10 mM DTT (Invitrogen), and 5U Recombinant RNasin® Ribonuclease Inhibitor (Promega) per well (53 (link)). The plate holder of the sorter was kept at 4°C throughout the sorting procedure. Random, negative and tetramer positive CD19⁺CD27⁺CD14⁻, CD3⁻, Hoechst⁻ B cells were sorted into 96-well plates, which were immediately put on dry ice and stored at −80°C.

PBMCs were stained immediately after thawing in FACS buffer (PBS with 0.1% NaN₃ and 2% FBS; Lonza). Color compensations were done using a mix of cells and compensation beads (BD Biosciences). After incubation of the cells with antibodies on ice for 30 min, they were washed with FACS buffer. Hoechst (Invitrogen) live/dead marker was added shortly prior to measuring on a FACSAria SORP machine (BD Biosciences). The following antibodies were used in the panel: CD14-eFluor605NC, CD24-eF450, CD43-APC, CD23-APC-eFluor780 (eBioscience), IgD-BV421, CD19-BV605, IgG-PE (BD Pharmingen), CD10-BV510, CD138-BV711, CD27-PECF594 (BD Horizon), IgM-BV570, CD38-PerCP-Cy5.5, CD21-PE-Cy7, CD20-AF700 (BioLegend), CD5-FITC, CD3-PE-Dy647 (Immunotools).