Bu 1 clone av20

Antibodies and dilutions used were as follows: chicken CD3 (clone CT3, Southern Biotech, Birmingham, Alabama, USA), 1:100 for FACS; Bu-1 (clone AV20, Southern Biotech), 1:1,000 for FACS, 1:300 for immunofluorescence; CD45 (clone AV53), 1:1,000 for FACS and immunofluorescence; chicken CSF1R [20 (link)], 1:100 for immunofluorescence; GFP Rabbit IgG Antibody Fraction, Alexa Fluor 488 Conjugate (Invitrogen, Carlsbad, CA, USA), 1:500 for immunofluorescence; Goat-anti-mouse-IgG1-alexa 647 (Invitrogen), 1:5,000 for FACS; Alexa Fluor 546 F(ab')2 Fragment of Goat Anti-Mouse IgG (Invitrogen), 1:500 for immunofluorescence.

Whole blood samples were incubated with the following labelled antibodies: CD45-APC (clone LT40, dilution 1:200), CD3-PE (clone CT-3, dilution 1:200), KUL01-FITC (dilution 1:50, Southern Biotech, Birmingham, AL, USA) and unlabeled antibodies: Bu-1 (clone AV20, dilution 1:500), MHC-II (clone 2G11, dilution 1:500) (Southern Biotech), CD41/CD61 (clone 11C3, dilution 1:100) and CD40 (clone AV79, dilution 1:100) (AbD Serotec, Kidlington, UK) for 30 min at 4 °C. After washing, samples with unlabeled antibodies were subsequently incubated for 30 min with secondary BV421 or PerCP-Cy5.5-labelled rat-anti-mouse antibodies (clone RMG1-1, dilution 1:500/1:800 respectively, BioLegend, San Diego, CA, USA). Samples were washed and analyzed by flow cytometry (FACSCantoII, BD Biosciences, San Jose, CA, USA) using FlowJo analysis software. Absolute cell numbers were determined using BD Trucount absolute counting tubes (BD Biosciences) according to the manufacturer’s protocol. Cell suspensions from cecal tonsils and spleen were also stained with above-mentioned antibodies and protocol.