Anti biotin microbeads and ms columns

Eosinophils were isolated from proximal SI LP by Percoll (described above) and by negative selection with anti-biotin MicroBeads and MS columns (Miltenyi Biotec) according to the manufacturer’s protocol. Cells were incubated with Fc block and biotinylated antibodies against the following antigens: IgA, MHC class II, Ly6g, CD115, B220, CD140a, CD90.2, and CD8a. Eosinophils were enriched to >90% purity. Approximately 50 × 10⁴ eosinophils per well were cultured in complete RPMI with 100 nM all-trans-RA (Sigma) or DMSO (vehicle control) alone. Eosinophil viability was measured by flow cytometry on day 3 of culture (% of eosinophils exhibiting weak staining with Zombie Yellow dye).

Cells from the peritoneal B-1 cell culture, submitted to conditions described in each experiment, were submitted to separation by selection in FACSAriaII (BD Biosciences). Cells in the non-adherent fraction were harvested, labeled with biotin-conjugated rat anti-mouse CD19 (BD Biosciences) and the CD19+ population enriched by using Anti-Biotin MicroBeads and MS Columns (Milteny Biotec) accordingly to the manufacturer instructions. Enrichment of ≥ 98% of CD19+ cells was set as the minimum acceptable experimental condition. The percentage of B-1 cell enrichment was verified by the addition of streptavidin-PE (BD Biosciences) and analysis in a FACS CantoII system (BD Biosciences).