Chicken Immune Cell Phenotyping

Samples from chickens in each group on each sample day were softly macerated, filtered through a 70-µm cell strainer (FALCON-Corning, NC, USA) into a centrifuge tube, and centrifuged at 352× g for 5 min. The cell pellets were suspended in 1 mL of PBS and counted; cells equivalent to 1 × 10⁶/mL from each sample were transferred to a Falcon tube (FALCON-Corning) and stained with Mouse Anti-Chicken CD3-FITC (Fluorescein isothiocyanate) [20 ], Mouse Anti-Chicken CD4-APC (Allophycocyanin) [21 (link)], and Mouse Anti-Chicken CD8-PE (Phycoerythrin) [20 ] antibodies (SouthernBiotech, Birmingham, AL, USA). The cells were then washed with phosphate-buffered saline (PBS) (PH7.4, 0.01 M, 4°C) 3× and suspended in 500 L of PBS for CD3+, CD4+, and CD8+ phenotyping using a BD FACS (Becton, Dickinson fluorescence-activated cell sorting) Calibur flow cytometer (BD Biotec., San Diego, CA, USA). The generated data were analyzed using Cell Quest software (BD Biotec.).