Flow cytometry was conducted to analyze the frequency of CD4+ cells, CD8+ cells,
interferon γ (IFN-γ), and the ratio of CD4+/CD8+. Cells were stained with 10 µL
of anti-porcine CD25 (AbD Serotec, MCA1736 clone K231.3B2), followed by 0.06 µg
of Alexa Fluor 488 goat anti-mouse immunoglobulin G (IgG, H+L; Invitrogen,
Molecular Probes, Carlsbad, CA, USA). The LYNX rapid antibody conjugation kit
(AbD Serotec) was used to conjugate mouse anti-porcine CD4 with allophycocyanin
(APC; clone 74-12-4; VMRD, Inc., Pullman, Wa, USA) and CD8 with RPECy7 (clone
76-2-11; VMRD, Inc.), and 0.05 µg of these antibodies were then added to the
cell preparation. Forkhead box P3 (Foxp3) intracellular staining was performed
with 0.03 µg of anti-rat/mouse Foxp3 (clone FJK-16s, with cross-reactivity with
swine Foxp3; eBioscience, San Diego, CA, USA) and 0.03 µg of PE-conjugated rat
IgG2a isotype control (clone eBR2a; eBioscience) using the Foxp3 Staining Buffer
Set (Staining, Fixation/Permeabilization, and Permeabilization Buffers;
eBioscience) to obtain the four-color stain
CD4APCCD8RPECy7CD25Alexa488Foxp3PE.
The frequency of regulatory T cells (Tregs) was evaluated by flow cytometry (BD
FACS Canto II), and data were analyzed using BD FACS Diva 6.0 software.
interferon γ (IFN-γ), and the ratio of CD4+/CD8+. Cells were stained with 10 µL
of anti-porcine CD25 (AbD Serotec, MCA1736 clone K231.3B2), followed by 0.06 µg
of Alexa Fluor 488 goat anti-mouse immunoglobulin G (IgG, H+L; Invitrogen,
Molecular Probes, Carlsbad, CA, USA). The LYNX rapid antibody conjugation kit
(AbD Serotec) was used to conjugate mouse anti-porcine CD4 with allophycocyanin
(APC; clone 74-12-4; VMRD, Inc., Pullman, Wa, USA) and CD8 with RPECy7 (clone
76-2-11; VMRD, Inc.), and 0.05 µg of these antibodies were then added to the
cell preparation. Forkhead box P3 (Foxp3) intracellular staining was performed
with 0.03 µg of anti-rat/mouse Foxp3 (clone FJK-16s, with cross-reactivity with
swine Foxp3; eBioscience, San Diego, CA, USA) and 0.03 µg of PE-conjugated rat
IgG2a isotype control (clone eBR2a; eBioscience) using the Foxp3 Staining Buffer
Set (Staining, Fixation/Permeabilization, and Permeabilization Buffers;
eBioscience) to obtain the four-color stain
CD4APCCD8RPECy7CD25Alexa488Foxp3PE.
The frequency of regulatory T cells (Tregs) was evaluated by flow cytometry (BD
FACS Canto II), and data were analyzed using BD FACS Diva 6.0 software.