Cd43 macs microbeads

Single cell suspensions of splenic cells were prepared. In some experiments red blood cells were lysed using ammonium chloride TRIS. In most experiments, live cells were isolated using Lympholyte M kit (Cedarlane, Burlington, Ontario, Canada) and B cells were purified by CD43 negative selection using MACS CD43-microbeads (Miltenyi Biotec Inc., San Diego, CA.). Resultant populations were routinely >97% B cells based on B220 staining and FACS analysis. Ex vivo B cells and splenocytes were cultured in IMDM supplemented with 10% FBS, sodium pyruvate (1 mM), L-glutamine (2 mM), 1% penicillin/ streptomycin, 2-ME (50 μM), HEPES buffer (10 mM), and 1% non-essential amino acids.
Ex vivo B cells or splenocytes were seeded (3 × 10⁵ cells/100 μL/well) in 96 well flat-bottom or U-bottom plates and stimulated with various concentrations of CDNs or IL-4 for the indicated time. The following stimuli were used: IL-4 (Supernatant from a J558L culture, 1:200 dilution); c-diGMP (C 057, Biolog life science institute, Federal Republic of Germany); 2′, 3′ cGAMP (c[G(2′,5′)pA(3′,5′)p], C 161, Biolog life science institute, Federal Republic of Germany); 3′, 3′ cGAMP (c-(ApGp), C 117, Biolog life science institute, Federal Republic of Germany); c-diAMP (C 088, Biolog life science institute, Federal Republic of Germany); Rp, Rp-c-diAMPSS (C118, Biolog life science institute, Federal Republic of Germany).

Based on their phenotypic characteristics, Breg^IL-33 cells were enriched and isolated from peripheral blood of IL-33-injected WT and IL-10^−/− mice for further functional characterization both in vitro and in vivo. Briefly, the mice were sacrificed at the peak of Breg^IL-33 appearing in the blood, i.e. week-2 after the 1st IL-33 injection (a total of 5 injections) (Fig. 3). Blood was harvested by heart puncture and diluted in PBS +5% EDTA. PBMC were first isolated by density separation using Lympholyte-Mammal medium (Cedarlane Laboratories Ltd, Canada). Total B cells (CD43^-) were then negatively selected using the MACS CD43 microbeads (Miltenyi Biotec, Germany), followed by depletion of CD23⁺ cells using an anti-CD23-PE antibody (Clone B3B4, Becton Dickinson, UK) and anti-PE microbeads (Miltenyi Biotec, Germany). All procedures were performed according to the manufacturers' instructions. Purities obtained were: total B cells (CD19⁺) >95%; of which the negatively selected population (CD23⁻ B cells) were >80%, and the remaining positively selected (CD23⁺ B cells) >90%. The flow through obtained after both depletion steps was enriched in CD23⁻ B cells with IL-10 producing capacity (∼3 folds), majority of which were also of CD25⁺ B cells (data not shown).

Bone marrow (BM) and spleens were harvested from four 8-week-old mice (two λ-MYC and two WT controls), and RNA was extracted from FACS-sorted BM populations (Pre-Pro-B [B220+CD19-], Pro-B [B220+CD19+IgD-IgM-CD25-], Pre-B [B220+CD19+IgD-IgM-CD25+], and immature B cells [B220+CD19+IgD-IgM+CD25-]) and from recirculating splenic B cells obtained by immunomagnetic negative selection with CD43 MACS Microbeads (Miltenyi Biotec-CD43 (Ly-48) Microbeads Mouse). cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and quantified by SYBR green assay (Applied Biosystems) with normalization to GAPDH expression. The following primers were used: GAPDH (forward) 5′-TGA AGC AGG CAT CTG AGG G-3′, (reverse) 5′-CGA AGG TGG AAG AGT GGG AG-3′; mouse c-Myc (forward) 5’ GAG CTC CTC GAG CTG TTT GA 3’, (reverse) 5’ AGT TCA CGT TGA GGG GCA TC 3’; human c-MYC (forward) 5’ GGA CCC GCT TCT CTG AAA GG 3’, (reverse) 5’ GAG GCT GCT GGT TTT CCA CTA 3’.