To determine CD3+, CD4+ and CD8+T cell
population, 200 μl of fresh whole blood collected in a K2EDTA tube
was stained with 15 μl of mouse anti-porcine CD3e-FITC (Southern Biotech,
Birmingham, AL), 5 μl PE-CYTM7 mouse anti-pig CD4a (BD Biosciences),
2.5 μl PE mouse anti-pig C8a (BD Biosciences, Franklin Lakes, NJ), and 20 μl
mouse anti-pig CD45: Alexa Fluor® 647 (Bio-Rad Antibodies, Hercules, CA).
Negative isotype controls recommended by the supplier for the previously
mentioned Abs were used in a separate tube. Abs were incubated at room
temperature for 15 min in a dark space protected from light. After the
incubation period, red blood cells were lysed twice with 2.7 ml deionized water
for 30 s and subsequent neutralization with 0.3 ml 10× PBS, followed by
centrifugation at 1000 g for 5 min at 22°C. Then cells were
washed and centrifuged twice with 2 ml 1× PBS. White blood cell (WBC) pellets
were resuspended in 1 ml of 1× PBS and data were collected by flow cytometry (BD
FACSCantoTMII, BD Biosciences) utilizing BD FACSDIVATMSoftware (BD Biosciences). Cell population was calculated by using AccuCount
Rainbow Fluorescent Particles (106 beads/ml, Spherotech, Lake Forest,
IL). Data acquisition was made until 1000 events of AccuCount beads were
collected. Lymphocyte population analyses and graphs were made with FLOWJOv10
software (Ashland, OR).
population, 200 μl of fresh whole blood collected in a K2EDTA tube
was stained with 15 μl of mouse anti-porcine CD3e-FITC (Southern Biotech,
Birmingham, AL), 5 μl PE-CYTM7 mouse anti-pig CD4a (BD Biosciences),
2.5 μl PE mouse anti-pig C8a (BD Biosciences, Franklin Lakes, NJ), and 20 μl
mouse anti-pig CD45: Alexa Fluor® 647 (Bio-Rad Antibodies, Hercules, CA).
Negative isotype controls recommended by the supplier for the previously
mentioned Abs were used in a separate tube. Abs were incubated at room
temperature for 15 min in a dark space protected from light. After the
incubation period, red blood cells were lysed twice with 2.7 ml deionized water
for 30 s and subsequent neutralization with 0.3 ml 10× PBS, followed by
centrifugation at 1000 g for 5 min at 22°C. Then cells were
washed and centrifuged twice with 2 ml 1× PBS. White blood cell (WBC) pellets
were resuspended in 1 ml of 1× PBS and data were collected by flow cytometry (BD
FACSCantoTMII, BD Biosciences) utilizing BD FACSDIVATMSoftware (BD Biosciences). Cell population was calculated by using AccuCount
Rainbow Fluorescent Particles (106 beads/ml, Spherotech, Lake Forest,
IL). Data acquisition was made until 1000 events of AccuCount beads were
collected. Lymphocyte population analyses and graphs were made with FLOWJOv10
software (Ashland, OR).