Rat anti mouse igm fitc

To assess cell surface BCR levels, 10⁶ B cells were resuspended in 50 μL ice-cold FACS buffer (PBS with 2% FCS), and then stained on ice for 30 min with Alexa Fluor 647-conjugated goat anti-human IgM Fab fragments (Jackson ImmunoResearch, #109-607-043, 1:200) or with rat anti-mouse IgM FITC (Invitrogen, #11-5790-81, 1:200) to detect the D1.3 BCR. Flow cytometry was performed using an LSRII-561 cytometer (Becton Dickinson Biosciences). Data were analyzed using FlowJo software (Treestar Inc.) using forward and side scatter to gate on single intact cells. Forward scatter was used as a relative measure of cell size.

The Ramos human IgM⁺ B cell line was obtained from ATCC (#CRL-1596). Raji D1.3 B cells, which express a transgenic hen egg lysozyme (HEL)-specific BCR were a gift from Dr. Bebhinn Treanor (University of Toronto, Toronto, Canada). A population of Raji D1.3 B cells with high expression of the D1.3 IgM-BCR was obtained by FACS sorting the cells after staining with rat anti-mouse IgM-FITC (Invitrogen, #11-5790-81, 1:200 dilution). Cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 1 mM sodium pyruvate, 2 mM glutamine, and 50 μM β-mercaptoethanol. All cells were confirmed to be mycoplasma-negative.