Mouse b cell isolation kit

Single cell suspension of splenocytes were generated from C57BL/6-Ly5.1 mouse spleen under sterile conditions by passing cells through a 70 μM cell strainer using the plunge of a syringe. Subsequently, cells were counted and pelleted at 300 g for 10 min at 4°C before resuspending them in autoMACS running buffer (Miltenyi Biotech). Highly pure resting B cells were isolated by magnetic labeling and depletion of CD43-expressing B cells and non-B cells using the Mouse B cell Isolation Kit (Miltenyi Biotech, 130-090-862) and MACS LS columns (Miltenyi Biotech) following vendor instructions. For activation, up to 3 × 10⁷ cells were plated in a T-75 flask on a 40 LB feeder cell layer for 24 h, if not described differently.

Human peripheral blood mononuclear cells were donated by the Chungbuk Red Cross Blood Center (Cheongju, Korea). Lymphocytes were isolated from these cells by density gradient centrifugation (Ficoll-Paque) 34 (link). hB cells were isolated from lymphocytes using a human B cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). mB cells were purified from spleen cells of MRL.Fas^lpr mice by a negative depletion method using mouse B cell isolation kit (Miltenyi Biotec) 9 (link). Purity of hB and mB cells was typically >90%. B cells (1 × 10⁵ cells/well) and MSCs (0.01-0.1 × 10⁵ cells/well) were added in 200 µL to the wells of 96-well plates. CpG-oligodeoxynucleotide (ODN; 5 μg/mL) was used to activate hB cells, and lipopolysaccharide (LPS; 1 μg/mL) was used to activate mB cells. To measure B cell proliferation, cells were pulsed with [³H]-thymidine (113 Ci/nmol; NEN, Boston, MA, USA) at a concentration of 1 μCi/well for the last 18 h and were harvested on day 3 using an automated cell harvester (Inotech, Dottikon, Switzerland). The amount of [³H]-thymidine incorporated into the cells was measured using a Wallac Microbeta scintillation counter (Wallac, Turku, Finland) 3 (link).

FV-specific and OVA-specific CD8 T cells were isolated from the spleens of female FV-specific CD8 TCR tg and OVA-specific TCR tg OT-1 mice using the BD IMag CD8 T Lymphocyte Enrichment Set (BD Pharmingen) according to the manufacturer’s instructions (purity >95% as determined by FACS). Splenic B cells were isolated from the spleen of female C57BL/6 wt, FcγRI^−/−, FcγRII^−/−, and FcγRIII^−/− mice using a mouse B cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Isolated B cell purity was more than 95%, as determined by flow cytometry.

Mouse spleens were homogenised through 40 μm cell strainers and red blood cells were lysed by incubation with Red Blood Cell Lysing Buffer (R7757, Sigma-Aldrich) for 5 min at room temperature. Naïve B cells were isolated by negative selection using a mouse B Cell Isolation Kit (130-090-862, Miltenyi Biotec) and LS magnetic columns (Miltenyi Biotec) according to the manufacturer's protocol.
Before BCR stimulation, purified naïve B cells were resuspended at 4 × 10⁶ cells/ml in 0.5% FBS RPMI media and rested for 75 min at 37°C. Cells were collected by centrifugation then incubated at 2 × 10⁷ cells/ml in 0.5% FBS RPMI containing 50 μg/mL goat anti-mouse IgM [F(ab′)₂ fragment; 115-036-075, Jackson ImmunoResearch] for 30 min on ice. B cells were then stimulated by incubation at 37°C for 2.5 min. Cells were immediately centrifuged at 800 × g for 2 min at 4°C and then resuspended in ice-cold lysis buffer (100 μl per 1 × 10⁷ cells), incubated on ice for 10 min and cleared by centrifugation at 20,000 × g for 10 min at 4°C.

Spleens were cut into small pieces and digested with collagenase type IV (Sigma, St. Louis, MO, USA). After hemolysis with ammonium chloride with potassium (ACK) lysis buffer, magnetic separation of B cells was performed using a Mouse B Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s protocol. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, BioWest, Nuaillé, France), 100 μg/ml l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA, USA), and 50 µM 2-mercaptoethanol (2-ME, Sigma).