Pe conjugated mouse anti chicken cd8

Cecal tonsil samples were collected from one bird/pen (n = 6) at d14 and d21 of age. For flow cytometry analysis, both treatment groups consisted of six samples, in duplicates, for each time point. Cecal tonsil samples were teased over a 0.4 μm cell strainer (Sigma, MO) with 2 mL RPMI-1640 media to obtain a single-cell suspension. Single-cell suspensions were concentrated for lymphocytes by density centrifugation over Histopaque (1.077 g/mL; Sigma, MO). For CD4⁺/CD8⁺ analysis, single-cell suspensions of the cecal tonsils (1 × 10⁶ cells) were incubated with FITC-conjugated mouse anti-chicken CD4, PE-conjugated mouse anti-chicken CD8 (Southern Biotech, Birmingham, AL) at 1:200 dilution, and unlabeled mouse IgG at 1:200 dilution in a 96-well plate for 20 minutes. After incubation, cells were washed twice to remove unbound primary antibodies at 400 ×g for 5 minutes using wash buffer (1× PBS, 2 mM EDTA, 1.5% FBS). After washing, cells were analyzed using cytosoft software (Guava Easycyte, Millipore, Billerica, MA). The CD4⁺ and CD8⁺ and CD4⁺/CD8⁺ cell percentages were analyzed after gating cells based on forward-scatter and side-scatter plot for lymphocytes.