Fortessa lsr2

Single-cell suspensions of CD43⁻ spleen cells of wt, ΔleftPAL and ΔIRIS mice (8–12 weeks old, male and female) were cultured for 3 days at 1 × 10⁶ cells per ml in RPMI 1640 with 10% fetal calf serum, 5 μg ml⁻¹ LPS with or without 20 ng ml⁻¹ IL-4, 2 ng ml⁻¹ TGFβ and 2 ng ml⁻¹ INFγ (PeproTech, Rocky Hill, NJ)6 (link)36 (link). At day 3, 1 × 10⁶ cells were cultured for 24 h in growth medium without LPS+cytokine. Supernatants were recovered and stored at −20 °C until use. At day 3, cultured splenic B cells were incubated with anti-B220-SpectralRed (PC5)-labelled antibodies (Biolegend, ref: 103212) and anti-IgG₁- (ref: 107020), anti-IgG_2a- (ref: 108002), anti-IgG_2b- (ref: 109002), anti-IgG₃- (ref: 110002) and anti-IgA- (ref: 104002) fluorescein-isothiocyanate (FITC)-labelled antibodies (Southern Biotechnologies), and then analysed on a Fortessa LSR2 (Beckton-Dickinson)6 (link). All antibodies were at a concentration of 10 μg ml⁻¹.

Cells were incubated with anti-B220-BV510, anti-CD21-BV650, anti-CD23-PC7, anti-IgM-APC, anti-IgMa-FITC, anti-IgMb-PE, anti-CD19-APCH7 and anti-AA4.1-APC antibodies (Southern Biotechnologies and Beckton Dickinson) and analysed on a Fortessa LSR2 (Beckton Dickinson) [19 (link)–21 (link)]. Heterozygous IgH a^Δ3′RR/b^wt mice generated by crossing homozygous 3′RR-deficient mice (IgH a^Δ3′RR/a^Δ3′RR) with C57BL/6 mice (IgH b^wt/b^wt) were investigated. Mixed Sv/129 × C57BL/6 mice (IgH a^wt/b^wt) were used as control mice.