Anti chicken cd3 fitc

To obtain splenic lymphocytes, five chickens from each group were respectively euthanized 1 week after the first immunization (week 3) and 1 week after booster immunization (week 4). The lymphocytes were then separated as described in 2.9. To analyze the percentages of CD4⁺ T lymphocyte subsets, obtained splenic lymphocytes (10⁶ cells suspended in 100 μl PBS) were dually stained with anti-chicken CD3-FITC (Southern Biotech, Birmingham, AL, USA) and CD4-APC (Southern Biotech) for 30 min at 4°C in the dark. As for the proportions of CD8⁺ T-lymphocyte subsets, 10⁶ splenic lymphocytes suspended in 100 μl PBS were dually stained with anti-chicken CD3-FITC and CD8-PE (Southern Biotech). After being washed thrice in PBS, lymphocytes were characterized by flow cytometry (Beckman Coulter Inc., Brea, CA, USA). Before cell sorting, fluorescence compensation was conducted using the fluorescence minus one (FMO) control. Each group consisted of five replications, and each replication was measured once.

The proportions of spleen T lymphocyte subpopulations of CD4+ and CD8+ from the vaccinated chickens were analyzed using flow cytometry technique. One week after the primary and booster vaccination, spleens were collected from five chickens of each group to prepare the spleen lymphocytes as described by Sasai et al. (2000) (link). The spleens were cut into scraps and gently pushed through a mesh screen and cells were washed with calcium and magnesium-free Hank’s balanced salt solution (HBSS). Lymphocytes were collected using a separation solution (TBD, Tianjin, China) according to the manufacturer’s protocol. Lymphocytes suspensions (1 × 10⁶ cells/ml) were incubated with anti-chicken CD3-FITC, anti-chicken CD3-FITC + anti-chicken CD8-PE, anti-chicken CD3-FITC + anti-chicken CD4-PE (SouthernBiotech, Birmingham, AL, United States) in the dark for 30 min at 4°C separately. Subsequently, the cells were washed twice with PBS by centrifugation (500 ×g, 10 min, 4°C) and re-suspended with cold PBS and analyzed with a BD FACSCalibur flow cytometer (BD, Franklin Lakes, NJ, United States). A lymphocyte specific gating was set according to forward and side scatter profiles. The proportions of CD4+ and CD8+ T lymphocytes were determined as described by (Song et al., 2010 (link)).