Mouse anti chicken cd8 pe

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).

Samples from chickens in each group on each sample day were softly macerated, filtered through a 70-µm cell strainer (FALCON-Corning, NC, USA) into a centrifuge tube, and centrifuged at 352× g for 5 min. The cell pellets were suspended in 1 mL of PBS and counted; cells equivalent to 1 × 10⁶/mL from each sample were transferred to a Falcon tube (FALCON-Corning) and stained with Mouse Anti-Chicken CD3-FITC (Fluorescein isothiocyanate) [20 ], Mouse Anti-Chicken CD4-APC (Allophycocyanin) [21 (link)], and Mouse Anti-Chicken CD8-PE (Phycoerythrin) [20 ] antibodies (SouthernBiotech, Birmingham, AL, USA). The cells were then washed with phosphate-buffered saline (PBS) (PH7.4, 0.01 M, 4°C) 3× and suspended in 500 L of PBS for CD3+, CD4+, and CD8+ phenotyping using a BD FACS (Becton, Dickinson fluorescence-activated cell sorting) Calibur flow cytometer (BD Biotec., San Diego, CA, USA). The generated data were analyzed using Cell Quest software (BD Biotec.).