Anti cd8 alexa fluor 647

To analyze the differentiation behavior of stimulated T cells, PBMCs stimulated with ConA were cocultured with naive or primed EVs at various concentrations (1, 5, and 10 μg/ml) for 3 days. After coculture, PBMCs were collected, washed with FACS buffer, and incubated with canine Fc receptor binding inhibitor on ice for 20 min. Then, PBMCs were stained with anti-CD3-FITC (Clone: CA17.2A12, Bio-Rad), anti-CD4-RPE (Clone: YKIX302.9, Bio-Rad), and anti-CD8-Alexa Fluor 647 (Clone: YCATE55.9, Bio-Rad) or their respective isotype controls. Fluorescence was evaluated by flow cytometry.

For immune cell population quantification, the 7 Holstein and 7 Jersey cows’ PBMCs were used for normal environmental conditions, and 8 Holstein and 7 Jersey cows’ PBMCs were used for high-heat environmental conditions using flow cytometry (FACS Canto II; BD Biosciences, Heidelberg, Germany). Briefly, PBMCs were stained with the following direct fluorescence-conjugated antibodies: anti-CD4:Alexa Fluor 647 (Bio-Rad, MCA1653A647), anti-CD8:Alexa Fluor 647 (Bio-Rad, MCA837A647), anti-CD11b:FITC (Bio-Rad, Hercules, California, USA; MCA1425F), anti-CD21:PE (Bio-Rad, MCA1424PE), anti-CD172a:PE-Cy5 (Bio-Rad, MCA2041C), anti-WC1:FITC (Bio-Rad, MCA838F), and anti-MHCII:FITC (Bio-Rad, MCA5656F). All conjugated antibodies were diluted 1:100 in PBS and incubated for 30 min in dark conditions. PBMCs were then fixed using 4 % paraformaldehyde (PFA) and stored at 4 °C until analysis. The flow cytometry analysis was conducted by three panel groups: (1) CD4, CD21, and WC1 antibodies for CD4 T cells, γδ T cells, and B cells; (2) CD11b and CD172a antibodies for monocytes; and (3) CD8 and MHCII antibodies for CD8 T cells and dendritic cells.