Mouse anti chicken bu 1 fitc clone av20

Mouse anti-chicken CD4-Alexa Fluor® 647 (clone CT-4, Cat. no. 821031), mouse anti-chicken CD8α-PE (clone 3-298, Cat. no. 8405–09), mouse anti-chicken CD3-SPRD (clone CT-3, Cat. no. 8200-13), and mouse anti-chicken Bu-1-FITC (clone AV20, Cat. no. 8395-02) antibodies were purchased from SouthernBiotech (Birmingham, AL, USA). The mouse anti-chicken monocyte/macrophage-PE antibody (clone 5K102, also called clone KUL01, Cat. no. M4520-17) was purchased from USBiological (Salem, MA, USA). Mouse anti-chicken MHC class II-FITC (clone 2G11, Cat. no. ab24882), mouse anti-chicken MHC class I-FITC (clone F21-2, Cat. no. ab24881), and biotin mouse anti-chicken TCR gamma delta (clone TCR1, Cat. no. ab25151) antibodies were purchased from Abcam. Streptavidin Alexa Fluor 750 conjugate (Cat. no. S21384) and Sytox blue dead cell stain (Cat. no. S34857) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The viability determination reagent 7-AAD (Cat. no. 8200-02), was purchased from BD Biosciences (San Jose, CA, USA).

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.