CD3+ (T cells) or B220+ (B cells) cells isolated (Miltenyi Biotec, Inc, per manufacturers’ directions) from WT or S5A splenocytes were incubated overnight with anti-CD3 (1 μg/ml; 145–2C11, eBioscience)/anti-CD28 (1 μg/ml; 37.51, eBioscience) (T cells) or plate-bound anti-IgM 10 μg/ml; F(ab’)2 fragment of goat anti-mouse IgM, Jackson ImmunoResearch, West Grove, PA) (B cells). Upregulation of CD69 and CD86 was assessed by flow cytometry (6 (link), 24 (link)).
For proliferation assays, CD3+ (T cells) or B220+ cells (B cells) isolated from WT or S5A splenocytes using negative selection (Miltenyi Biotec Inc.) and were labeled with CTV (Invitrogen). Cells were incubated for 72 h in the absence or presence of anti-CD3/anti-CD28 (T cells) or of soluble anti-IgM stimulation (10 μg/ml; F(ab’)2 fragment of goat anti-mouse IgM, Jackson ImmunoResearch, West Grove, PA), and rIL-4 (10 ng/ml, R&D Systems) (B cells). Cells were analyzed for CTV dilution by flow cytometry.
For proliferation assays, CD3+ (T cells) or B220+ cells (B cells) isolated from WT or S5A splenocytes using negative selection (Miltenyi Biotec Inc.) and were labeled with CTV (Invitrogen). Cells were incubated for 72 h in the absence or presence of anti-CD3/anti-CD28 (T cells) or of soluble anti-IgM stimulation (10 μg/ml; F(ab’)2 fragment of goat anti-mouse IgM, Jackson ImmunoResearch, West Grove, PA), and rIL-4 (10 ng/ml, R&D Systems) (B cells). Cells were analyzed for CTV dilution by flow cytometry.
