Cy5 pe labeled anti mouse b220

Splenic mouse B cells were isolated from mice by immunomagnetic depletion with anti-CD43 MicroBeads (Invitrogen) as previously described^{9 (link)}. Briefly, all the non-B cells were depleted with anti-CD43 MicroBeads combined with Dynabeads Biotin Binder (Invitrogen); naive B cells were cultured at a concentration of 5×10⁵ cells/ml in RPMI medium supplemented with 15% FBS, penicillin-streptomycin (100 units/ml), L-Glutamine (2 mM), anti-CD40 (1 μg/ml, eBioscience) and recombinant mouse IL-4 (20 ng/ml; PeproTech). The purity of B cell population was typically 96–98% in all experiments, as documented by flow cytometric analysis of B220 expression in enriched cells. Cells were collected after 4 days of treatment with inhibitors to isolate genomic DNA for HTGTS libraries and targeted re-sequencing experiments. For RNA and protein extraction cells were collected at the indicated time points. Class switch recombination (CSR) was measured by staining with PE-labeled anti-mouse IgG₁ (BD Biosciences) and Cy5-PE-labeled anti-mouse B220 (eBiosciences). Data acquisition was performed using a FACSVerse flow cytometer (BD biosciences).

For immunization, sheep blood Alsevers (BD) were washed with PBS and re-suspended in PBS at a concentration of 1×10⁹ sheep red blood cells (SRBCs) per ml. 8 to 12-week old mice were immunized by intraperitoneal injection of 2×10⁸ SRBCs in a 200 ml volume. After 5 days, a booster injection was performed using 5-fold more SRBCs. On day 6 and for 7 consecutive days, animals were daily administered vehicle (0.5 % carboxymethylcellulose, 0.05 % Tween 80 in ultra-pure water) or idelalisib or duvelisib (10 mg/kg q.d.) by oral gavage. Spleens were collected at the end of treatment, placed on ice, washed in PBS to remove residual blood, cut into small pieces, crushed and physically dissociated using a Falcon cell strainer, and subjected to hypotonic lysis of erythrocytes. Mouse germinal center (GC) B cells were isolated from the spleens of immunized mice by immunomagnetic depletion: first non-B cells were depleted with anti-CD43 MicroBeads; next enriched B cells were incubated with a cocktail of biotinylated antibodies specific for CD11c (eBiosciences) and IgD (eBiosciences) to remove dendritic cells and mature naive B cells, respectively, as previously reported^{32 (link)}. Enrichment of the GC B cells was evaluated with PE-labeled anti-mouse GL7 (eBiosciences) and Cy5-PE-labeled anti-mouse B220 (eBiosciences).