Thymus and spleen tissues were prepared to obtain single-cell suspensions (2 × 106 cells) as previously described [48 (link)]. Immunofluorescence staining was performed with anti-chicken directly-conjugated MAbs: anti-CD4 FITC, anti-CD8 PE and anti-chT1 FITC (AbD Serotec, Ltd., Oxford, UK). Cell suspensions were added to PBS wash buffer containing 2 % bovine serum albumin and diluted antibodies for 30 min at 4 °C, followed by washing and resuspension in PBS containing 1 % paraformaldehyde. All cell populations were assessed using a FACSAria III (BD Biosciences, San Jose, CA, USA).
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