Mouse anti chicken cd3 fitc

On the 7th day after each immunization, five chickens in each group were dissected, and their spleens were removed. The spleens were well ground in PBS buffer and were filtered with a 200-mesh cell sieve; the filtrate was slowly added along the wall into a 10 mL sharp-bottomed glass centrifuge tube containing 5 mL of 37 °C pre-warmed lymphocyte separation solution (TBDscience, Tianjin, China) and was then centrifuged at 720 g for 16 min; the middle white layer of the cells was transferred into new centrifuge tubes and were washed twice with PBS buffer. Finally, the lymphocytes were counted using a blood counting chamber, and the density was adjusted to 1 × 10⁷ cell/mL by PBS buffer. An amount of 100 μL of counted lymphocytes were taken from each group and were placed into 2 mL centrifuge tubes; lymphocytes from all of the groups were double stained with mouse anti-chicken CD3-FITC (Southernbiotech, Birmingham, AL, USA) and mouse anti-chicken CD4-PE or mouse anti-chicken CD8-PE by incubating at 4 °C for 45 min under dark conditions; in addition, lymphocytes from the PBS control group were treated with blank or single stain for template adjustment. The sample test was run using a FACScan flow cytometer (BD Biosciences).

Three tissue samples of the liver, spleen, and thymus from each chicken group were used for immunophenotyping. A small piece of each tissue was softly macerated and filtered using a 70 μm cell strainer (FALCON-Corning, NC, USA) in a centrifuge tube and centrifuged at 376 × g for 5 min. The supernatant was discarded, and the cells were suspended in 1 mL PBS and mixed by gentle rocking, counted, and cells equivalent to 1 × 10⁶/mL from each sample were transferred to falcon tubes (FALCON-Corning) for staining. Mouse Anti-Chicken CD3-FITC [26 ], Mouse Anti-Chicken CD4-APC [27 (link)], and Mouse Anti-Chicken CD8α-PE [26 ] antibodies (SouthernBiotech, Birmingham, AL, USA) reconstituted according to the manufacturer’s recommendations were used for staining the prepared cells. Forty microliters of each antibody adequate for staining 1 × 10⁶ cells were transferred into each test falcon tube containing prepared cells and incubated at 4°C for 30 min. Then, the cells were washed with PBS (pH 7.4, 0.01 M, 4°C) thrice by centrifuging at 376 × g for 5 min at 4°C each. After washing, the cells were suspended in 500 μL PBS, sorted, assessed, and CD3+, CD4+, and CD8+ phenotypes were determined using flow cytometry on 10,000 live cells using a BD FACSCalibur flow cytometer (BD Biotec., San Diego, CA, USA) [28 (link)]. The data generated were analyzed using the CellQuest software (BD Biotec.).