Pe mouse anti pig cd4a

The collected PBMCs were washed in PBS and incubated for 15 min with FITC mouse anti-Pig CD3 (BD Biosciences, 559582) and PE mouse anti-Pig CD4a (BD Biosciences, 559586), according to the manufacturer’s protocol. PK-15 cells after a viral infection or drug treatment were collected using trypsin-EDTA and washed in PBS. The cells were resuspended in annexin buffer and incubated for 15 min with 2.5  µg/mL PI (BD Biosciences) and Annexin V (BD Biosciences), according to the manufacturer’s protocol. The antibody-labeled or necrotic cells were analyzed using the flow cytometer (Beckman Coulter Life Science, CytoFLEX). Flow cytometry analyses were reproduced by three independent experiments.

Porcine peripheral blood CD4⁺T cells were purified from PBMCs by immunomagnetic labeling of cells using mouse anti-pig CD4a monoclonal antibody (AbD Serotec) and anti-mouse IgG MicroBeads (Miltenyi Biotec). Cells were stained with PE mouse anti-pig CD4a (BD Biosciences); after washing, cells were re-suspended in sorting buffer for FACS analysis. Flow cytometry was performed using a FACSCalibur (BD Biosciences), and the purity of CD4⁺ T cells was demonstrated to be 98%.

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.