Bu 1 av20

Cells isolated from the spleen, liver, and intestinal epithelium were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain kit (L3457; Thermo Fisher, USA) and the following fluorescent antibodies to chicken CD3 (CT-3, Southern Biotech, USA), CD4 (CT-4, Southern Biotech, USA), CD8α (CT-8, Southern Biotech, USA), CD44 (AV6, Southern Biotech, USA), TCRγδ (TCR-1, Southern Biotech, USA), monocytes/macrophages (KUL01, Southern Biotech, USA), MHCII (2G11, Southern Biotech, USA), Bu-1 (AV20, Southern Biotech, USA), and CD45 (LT-40, Southern Biotech, USA). Stained cells were analyzed using a CytoFLEX flow cytometry and the data were analyzed using CytExpert Software (both from Beckman Coulter, USA).

PBMCs were isolated by Ficoll (Sigma Aldrich) density gradient centrifugation from EDTA‐treated blood or splenocyte cell suspensions. The following mouse‐anti‐chicken antibodies were used: Bu‐1 (AV20, conjugated or unconjugated), lambda‐PE (L‐1), TCR gamma/delta (TCR1), TCRαβ/Vβ1 (TCR2), TCR αβ/Vβ2 (TCR3), monocytes/macrophage marker (KUL01), Cμ‐CH1 (M1, conjugated or unconjugated), CD4 (CT4), and CD8 (CT8) (all from Southern Biotech), ms‐anti‐chicken‐Cμ (BK) made by Bernd Kaspers (binds a part of the constant region of Cμ other than CH1), or goat‐anti‐chicken‐IgM (Bethyl Laboratories). Secondary antibodies were: goat‐anti‐mouse‐IgG‐Alexa‐647 (Rockland), goat‐anti‐mouse‐IgG‐FITC (Thermo Fisher Scientific), or donkey‐anti‐goat‐IgG‐Alexa‐647 (Rockland). Fluorescence was measured using an Attune^TM Flow Cytometer (Thermo Fisher Scientific).