Fitc hamster anti mouse cd3e

Flow cytometry was performed on a CytoFLEX (Beckman), and data were analyzed by FlowJo software. The live cells were observed by the Fixable Viability Stain 780 (Cat#565388, BD Pharmingen). For cell-surface staining, cells were stained with BV510 Rat Anti-Mouse CD45(Cat#563891, BD Pharmingen), FITC Hamster Anti-Mouse CD3e (Cat#553061, BD Pharmingen), PE-Cy7 Rat Anti-Mouse CD4 (Cat#552775, BD Pharmingen), BV421 Rat Anti-Mouse CD25 (Cat#562606, BD Pharmingen), FITC Rat Anti-CD11b (Cat#557396, BD Pharmingen), BV421 Rat Anti-Mouse F4/80 (Cat#565411, BD Pharmingen), PE Rat Anti-Mouse CD86(Cat#561963 BD Pharmingen), Alexa Fluor 647 Rat Anti-Mouse CD206 (Cat#565250 BD Pharmingen). For intracellular staining, cells were stained with PE Rat Anti-Mouse IL-17A (Cat#561020 BD Pharmingen), Alexa Fluor 647 Rat anti-Mouse Foxp3(Cat#560402 BD Pharmingen). According to the reagent vendor’s instructions, Purified Rat Anti-Mouse CD16/CD32 is used to exclude background fluorescent signal interference, Cells fixation and permeabilization Fixation/Permeablization Kit (Cat#554714 BD Pharmingen) and Transcription Factor Buffer Set (Cat#562574 BD Pharmingen) were used for cell fixation and permeabilization.

At 28 dpv, the splenic lymphocytes of mice or the peripheral blood lymphocytes of piglets were isolated, transferred into a 1.5-mL centrifuge tube (1 × 10⁶ cells/tube), and washed once with PBS. The pellet was resuspended in 300 µL of cell fluorescence solution (Flow Cytometry Staining Buffer, eBioscience, USA) and stained with the following fluorescent antibodies: FITC hamster anti-mouse CD3e (BD Biosciences, USA), PE anti-mouse CD4 (BioLegend, USA), APC anti-mouse CD8a (BioLegend), FITC mouse anti-pig CD3ε (BD Biosciences), PE mouse anti-pig CD4a (BD Biosciences), and APC mouse anti-pig CD8α (SouthernBiotech, USA), at room temperature in the dark for 30 min. The tubes were centrifuged at 1,500 rpm for 5 min; the supernatant was removed, and the pellet was washed twice with PBS. The cell pellet was resuspended in 500 µL of fluorescence preservation solution (0.15 M PBS pH 7.4, 2% glucose, 1% formaldehyde, and 0.1% NaN₃). Flow cytometry, performed on a Accuri C6 Plus instrument (BD Biosciences), was then used to enumerate CD4⁺ and CD8⁺ T cells per 1 × 10⁵ cells acquired. Data analysis was performed using FlowJo v10.7.1.

At 21 and 42 days of age, splenic samples of eight mice in each group were taken to determine the percentages of CD3⁺, CD4⁺, CD8⁺ T lymphocyte and CD 19⁺ B lymphocyte by FCM.
Each spleen was cut into pieces and then filtered through nylon gauze as splenic single-cell suspension. The suspension was centrifuged at 200 × g for 5 min. The supernatant was discarded and lymphocytes were collected. The cell concentration was determined by using the normal counting method of blood cells and then diluted to 1.0 × 10⁶ cells/mL with phosphate-buffered saline (PBS). A total of 100 μL cell suspensions was transferred to another centrifuge tube. The cells were respectively stained with 10 μL hamster anti-mouse CD3e-FITC (BD, Cat No: 553062), rat anti-mouse CD4-PE (BD, Cat No: 557308), rat anti-mouse CD8a-PerCP (BD, Cat No: 553036) and FITC anti-mouse CD19⁺(BD, Cat No: 553785) for 30 min at RT, and then 2 mL PBS was added and centrifuged at 200 × g for 5 min. The supernatant was discarded. Cells were resuspended in 0.5 mL PBS and determined by BD FACS Calibur flow cytometer.

At 21 and 42 days, the blood samples were taken from retro-ocular artery. The peripheral blood of eight mice in each group were taken to measure the percentages of CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺ T lymphocyte by flow cytometry.
100 μL anti-clotting peripheral blood was put in a test tube. And then added 10 μL hamster anti-mouse CD3e-FITC (BD, Cat No: 553062), rat anti-mouse CD4-PE (BD, Cat No: 557308) and rat anti-mouse CD8a-PerCP (BD, Cat No: 553036) to the test tube for 30 min at RT, and then 1 mL lysing solution (BD, Cat No: 349202) was added for 10 min at room temperature. Subsequently, 1 mL PBS was added and centrifuged at 200×g for 5 min. The supernatant was discarded. 2 mL PBS was added and centrifuged at 200×g for 5 min and the supernatant was discarded. The cells were resuspended in 0.5 mL PBS and determined by BD FACS Calibur flow cytomyter.