Mouse anti chicken monocyte macrophage pe clone kul01

Primary cells were isolated from the BF of an additional five uninfected line W and an additional five uninfected line 15 birds as described. Cells were re-suspended in FACS buffer (1% bovine serum albumin (BSA) in 1× PBS), plated in U-bottom 96-well plates and treated with 4% BSA for 20 min at room temperature (RT) to block the Fc receptors. Cells were then surface stained with mouse anti-chicken Bu-1-FITC (Clone AV20; Southern Biotech, Birmingham, AL, USA) and mouse anti-chicken monocyte/macrophage-PE (Clone KUL01, Southern Biotech) antibodies in the dark for 20 min at RT. Finally, the cells were fixed in 4% paraformaldehyde, re-suspended in 400 µL FACS buffer and subject to flow cytometry using BD LSRFortessaTM Flow cytometer (BD Biosciences, San Jose, CA, USA). Ten thousand events were collected per sample, and gated based on forward scatter (FSC) and side scatter (SSC). Data were analysed using FlowJo software (FlowJo_v_10.6.2, Ashland, OR, USA) to count the percentage of Bu1+ and KUL01+ cells in the BF population.