Fitc conjugated anti chicken cd3

As described in section 2.8, splenic lymphocytes were collected and resuspended in 1 × PBS. To evaluate the proportions of CD4⁺ T lymphocytes subsets, 100 μl medium containing 10⁶ lymphocytes were dually stained with FITC-conjugated anti-chicken CD3 (Southern Biotech, Birmingham, AL, USA) and APC-conjugated anti-chicken CD4 (Southern Biotech, Birmingham, AL, USA) for 30 min at 4°C in the dark. As for the percentages of CD8⁺ T lymphocytes subsets, 10⁶ lymphocytes were suspended in 100 μl PBS, and stained with FITC-conjugated anti-chicken CD3 and PE-conjugated anti-chicken CD8 (Southern Biotech, Birmingham, AL, USA) by the same strategy. After being washed in 1 × PBS, cells were sorted by a flow cytometry (Beckman Coulter Inc, Brea, CA, USA), and the populations were determined by CytExpert software (Beckman Coulter Inc, Brea, CA, USA). Noticeably, fluorescence compensation was performed based on the fluorescence minus one (FMO) control before cell sorting. Each group involved five biological replicates, and each replication was detected once.

Single-cell suspensions from blood were prepared using a peripheral-blood lymphocyte separation kit (Solarbio, Beijing, China), and flow cytometry was performed to detect the percentages of CD3⁺CD4⁺, CD3⁺CD8⁺ T-cells [57 (link)]. The isolated cells (1 × 10⁶) were incubated with FITC-conjugated anti-chicken CD3 and PE-conjugated anti-chicken CD4 or CD8 (SouthernBiotech) monoclonal antibodies, respectively. The samples were quantified by a flow cytometer (BD LSRFortessa™, USA). Experiments were performed in triplicates and the data were analyzed using FlowJo (Version 7.6.2, Becton, Dickinson and Company, USA).