Anti mouse iga pe

In vitro IgM to IgA class switching was measured by flow cytometry. CH12F3 cells (10 000 cells/ml) were seeded and stimulated in flat-bottomed 96-well culture plates in 200 μl growth medium containing 2 μg/ml hamster anti-mouse CD40 (BD Biosciences), 10 ng/ml recombinant murine IL-4 (Peprotech) and 1 ng/ml human recombinant TGF-β1 (PeproTech) (+CIT). Cells were harvested after 72, 96 or 120 h and stained with LIVE/DEAD red stain (Invitrogen), blocked with Fc receptor antibody (2.4G2), fixed and permeabilised in CytoFix/Cytoperm™ and washed in PermWash™ containing saponin. IgA was stained using anti-mouse IgA-PE (eBioscience, 1:200). Cells were washed twice with PermWash™ and suspended in 200 μl PBS before analysis on a FACSCanto™ (BD Biosciences). Viable CH12F3 cells were analysed for IgA expression using FlowJo^® version 10 software. Reagents were from BD Biosciences if not stated otherwise.

In vitro IgM to IgA class switching was measured using flow cytometry. CH12F3 cells (10 000 cells/ml) were seeded in flat-bottomed 96-well culture dishes in 200 μl growth medium. Cells were stimulated with 2 μg/ml hamster anti-mouse CD40 (BD Biosciences) and 10 ng/ml recombinant murine IL-4 (Peprotech), and 1 ng/ml human recombinant TGF-β1 (PeproTech) for 4 days. The cells were then stained with LIVE/DEAD red stain (Invitrogen), blocked with Fc receptor antibody (2.4G2) and normal mouse serum (Invitrogen), fixed and permeabilized in CytoFix/CytopermTM and washed in PermWashTM containing saponin. IgA was stained using anti-mouse IgA-PE (eBioscience, 1:200). Cells were washed twice with PermWashTM and suspended in 200 μl of CellFixTM before analysis on a FACS Canto. Viable CH12F3 cells were analyzed for IgA expression using FlowJo® version 10 software. Reagents were from BD Biosciences if not stated otherwise.