Cd19 bv605

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).

The following fluorochrome-conjugated anti-human antibodies and fluorescent dyes were used for flow cytometry analyses: CD19-BV605, CD3-BV421, and CD23-BV421 (BD Biosciences, Franklin Lakes, NJ, USA); IgM-APC (Biolegend, San Diego, CA, USA); and CD19-eVolve 605, CD38-PE-eFluor 610, CD24-APC-eFluor 780, CD27-APC, CD21-PerCP-eFluor710, IgD-PE, CD5-APC, 7AAD, Fixable Viability Dye eFluor 506, Propidium Iodide, Rhodamine 123 (R123) (Thermo Fisher Scientific). Resiquimod (R848) and glucopyranosyl lipid A (GLA) were obtained from the Infectious Disease Research Institute (Seattle, WA). C12-iE-DAP (iE-DAP), polyinosinic-polycytidylic (Poly I:C), trehalose-6,6-dibehenate (TDB), and CpG ODN 2006 (CpG) were purchased from InvivoGen (San Diego, CA).

Single cell suspensions extracted from the lungs were stained with a live/dead-Infrared marker (Thermo Fisher) and incubated with monoclonal antibody CD16/CD32 Becton Dickinson Biosciences (BD) to block Fc receptors, before adding a cocktail of directly conjugated mAbs directed against the following surface markers; CD11b-BV711 (M1/70, 563168), Ly6G-PerCp-Cy5.5 (1A8, 560602), CD45-BB515 (30-F11, 564590), SiglecF-PE-CF594 (E50-2440, 562757), CD103-BV421 (M290, 562771), CD11c-BV510 (HL3, 562949), CD3e-BV786 (145-2C11, 564379), CD19-BV605 (1D3, 563148), CD335-BV605 (29A1.4, 560469), and Ly6C-APC (AL-21, 560595) all purchased from BD and FceR1-PE-Cy7 (MAR-1, 25-5898-82), MHC-II-AF700 (M5/114.15.2, 56-5321-82) from Thermo Fisher (ebioscience). Data acquisition was performed with the BD LSR Fortessa (BD) and Diva software, and the analysis was performed with FlowJO X software (TreeStar, Ashland, OR). In initial control experiments, the right lobes were evaluated for the frequency of contaminating peripheral blood mononuclear cells after cardiac PBS infusion by staining with an anti-CD115 mAb (T38-320, 565249, BD), which is uniformly expressed on all circulating blood monocytes (13 (link)) but not on lung monocytes (14 (link)). The frequency of CD115⁺ cells was below 0.01% among the lung cells, arguing that we had little if any blood leukocyte contamination (Figure S1B).

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.

All antibodies are from BioLegend (San Diego) unless otherwise noted: CD16-APC-fire750, clone: 3G8; CD56-AF647, clone: NCAM; CD45RA-BV650, clone: HI100; CCR7-Pe/Dazzle 594, clone: G043H7; CD8-PeCy7, clone: SK1; CD45-A488, clone: SD1; CD4-AF700, clone: OKT4; CD19-BV605, clone: HIB19; CD3-BV785, clone: OKT3; HLADR-PE, clone: L243 (BD Biosciences, CA); CD14-PerCP-Cy5.5, clone: MφP9 (BD Biosciences, CA); Zombie Aqua™ Fixable Viability Kit.