Anti cd23 microbeads

Nine-week-old mice were euthanized, and BM cells were collected through flushing the femoral shafts with cold RPMI supplemented with 2% FBS and 2 mM EDTA (IBI Scientific, Dubuque, IA, USA). Red blood cells were depleted with a lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) and incubated with anti-CD23 MicroBeads; mature recirculating B cells were removed with the magnetically activated cell-sorting (MACS) system (Miltenyi Biotec) through positive selection using LS columns (Miltenyi Biotec). The negative fraction was recovered and subsequently incubated with anti-B220 MicroBeads (Miltenyi Biotec), so that B220+CD23- BM cells were purified by positive selection.

Total peritoneal cells from mice stimulated or not with L. amazonensis promastigotes or EVs released by the parasite were subjected to centrifugation at 161 × g for 5 min. The pellets containing the cells were resuspended in PBS pH 7.2 supplemented with 0.5% FBS, and 2 mM ethylenediaminetetraacetic acid—EDTA (MACS buffer) and the cells were counted in a Neubauer chamber. For enrichment 90 µl of MACS were added to every 10⁷ total cells. Cell suspensions were incubated with 2 μl of Fc block per 6 × 10⁶ total cells for 60 min at 4°C. Then, cells were washed with sterile PBS, resuspended in MACS, and subjected to negative selection with anti-CD23 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) followed by positive selection with anti-CD19 microbeads (Miltenyi Biotec) (CD23^- CD19⁺). All procedures were performed according to the manufacturer’s instructions. The purity of the cell suspension was evaluated by flow cytometry using anti-IgM APC (II/41 clone, BD) and anti-CD11b PE (M1/70 clone, BD) cell surface markers. The data were acquired in the FACSCalibur cytometer (BD Bioscience) and analyzed with FlowJo software version 10.6.2 (FlowJo, LLC).

Total peritoneal cells were obtained from a lavage of the peritoneal cavities of BALB/c mice. The total cells collected were subjected to magnetic cell sorting to obtain an enriched culture of B-1 cells by using negative selection with anti-CD23 microbeads (Miltenyi Biotec) and positive selection with anti-CD19 microbeads (Miltenyi Biotec) Gladbach, Germany) (e Brito et al., 2007 (link); Geraldo et al., 2016 (link)). Purified B-1 cells were labeled with anti-CD11b monoclonal antibodies conjugated to phycoerythrin (PE) and anti-IgM conjugated to fluorescein isothiocyanate (FITC) (all from BD, San Diego, CA, United States) and analyzed on a FACSCalibur flow cytometer (BD) to evaluate cell purity.