Rat anti mouse b220 ra3 6b2

FITC-labeled anti-mouse CD3 (145-2C11, #11-0031-82, 1:100) and GL7 (GL7, #13-5902-81, 1:200) were purchased from eBiosciences (San Diego, CA). Rat anti-mouse B220 (RA3-6B2, #550286, 1:100) and rat anti-mouse IgD (11-26c.2a, #553438, 1:100) were purchased from BD Biosciences (San Jose, USA). Biotinylated PNA (#B-1075,1:300), goat anti-mouse IgM (#FI-2020,1:100) and AMCA-labeled streptavidin (#SA-5008,1:300), biotinylated ulex europaeus agglutinin-1 (UEA-1, #B1065, 1:200) and FITC-labeled streptavidin (#SA-5001, 1:100) were purchased from Vector Laboratories (Burlingame, USA). Texas red-labeled goat anti-rat IgG (#T-6392, 1:500), Alexa Fluor^® 488-labeled goat anti-rat IgG (#A-11006, 1:500), and Alexa Fluor^® 488-labeled goat anti-mouse IgG (#A11001, 1:200) were purchased from Thermo Fisher Scientific (Waltham, USA). Rat anti-mouse cytokeratin 8 (#Troma-1 1:100) was purchased from Developmental Studies Hybridoma Bank (Iowa City, USA).

A mouse LP dissociation kit (Miltenyi Biotec) was used to isolate cells from the small intestine according to the manufacturer's protocol, with a modification at the final dissociation step where cells were gently crushed through a 70 μm cell strainer immediately following enzymatic digestion to generate a single cell suspension. Cells were centrifuged for 10 min at 1,500 rpm and the cell pellet was resuspended in PBS containing 0.5% BSA. Cells were counted using a hemocytometer prior to staining. Cells were stained with fixable viability dye eFluor506 (ThermoFisher) and Fc receptors were blocked with anti-mouse CD16/32 (2.4G2; BD Biosciences). Surface staining included rat anti-mouse CD45 (30-F11; BD Biosciences), rat anti-mouse B220 (RA3-6B2; BD Biosciences) and rat anti-mouse Siglec F (E50-2440; BD Biosciences). Cells were fixed and permeabilized using BD CytoFix/CytoPerm (BD Biosciences) and incubated with rat anti-mouse IgA (C10-1; BD Biosciences). Liquid counting beads (BD Biosciences) were added to samples prior to acquiring data on a CytoFLEX flow cytometer (Beckman Coulter) to enable total cell counts of cells falling within IgA⁺B220⁻CD45⁺ live gates according to manufacturer's protocol. Data were analyzed using CytExpert software (Beckman Coulter).