Blood samples were treated with ACK buffer containing 8.26 mg/mL of NH4Cl, 1.19 mg/mL of NaHCO3, and 37.8 mg/mL of 2Na-EDTA (pH 7.3), and then washed twice with PBS. The staining of Tregs was then performed as described previously (23 (link)). Briefly, the cells were stained using the following reagents: FITC-conjugated anti-bovine CD4 antibody (CC8; Bio-Rad), Alexa Fluor 647-labeled anti-bovine CD25 antibody (IL-A111; Bio-Rad), FOXP3 Fix/Perm Buffer (BioLegend, San Diego, CA, USA), and PerCP/Cy5.5-conjugated anti-bovine Foxp3 antibody (FJK-16s; eBioscience, San Diego, CA, USA). PerCP/Cy5.5-conjugated rat IgG2a isotype control (eBR2a; eBioscience) was used as a negative control. After staining, the cells were analyzed by FACS Verse (BD Biosciences).
CD69 staining was performed as described in a previous paper (32 (link)). Briefly, collected cells were stained using the following antibodies: PerCP/Cy5.5-conjugated anti-bovine CD3 antibody (MM1A; Washington State University Monoclonal Antibody Center, Pullman, WA, USA), FITC-conjugated anti-bovine CD4 antibody (CC8), PE-conjugated anti-bovine CD8 antibody (CC63; Bio-Rad), and Alexa Fluor 647-labeled anti-bovine CD69 antibody (KTSN7A; Kingfisher Biotech). After staining, the cells were analyzed by FACS Verse.
CD69 staining was performed as described in a previous paper (32 (link)). Briefly, collected cells were stained using the following antibodies: PerCP/Cy5.5-conjugated anti-bovine CD3 antibody (MM1A; Washington State University Monoclonal Antibody Center, Pullman, WA, USA), FITC-conjugated anti-bovine CD4 antibody (CC8), PE-conjugated anti-bovine CD8 antibody (CC63; Bio-Rad), and Alexa Fluor 647-labeled anti-bovine CD69 antibody (KTSN7A; Kingfisher Biotech). After staining, the cells were analyzed by FACS Verse.