T Cell Responses to Various Levels of Exercise Stress

Blood samples were collected using standard venipuncture technique. Blood samples were obtained at baseline, immediately post-exercise (0h), 1 hour post (1h), 4 hours (4h), and 6 hours (6h) post-exercise in heparin vacutainer tubes. Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Paque PLUS density gradient centrifugation. Total T cells were purified from washed PBMC by E-rosetting. T cell proliferation was analyzed in response to co-stimulation through CD3+CD28 using plate-bound antibodies, or phytohaemagglutinin (PHA) treatment, or no simulation. Cells were incubated for 6 days at 37˚C in a humidified incubator with 5% CO2 and then analyzed by flow cytometry. Cells were analyzed by flow cytometry using anti-CD3-APC, and anti-CD152-PE antibodies immediately after CD3+ T cell isolation (0 h) and following 6 d in culture using an Accuri C6 flow cytometer.

Blood will be collected in a vial containing EDTA (6 ml) for analysis of catecholamines. Plasma for catecholamine (epinephrine and norepinephrine) analyses was obtained by centrifugation at 2000 g for 10 min at 4˚C. The supernatant was removed and stored at -80˚C until analysis. Catecholamine concentrations will be determined using BI-CAT ELISA kits and performed per the manufacturer's instructions. The samples were measured at 450 nm using a Synergy HT microplate reader. The blood samples will be obtained at the same time as for the primary outcomes (prior to and following exercise as well as one, four and six hours post-exercise.

Peripheral blood will be collected in a vial containing EDTA (6 ml) for analysis of complete blood counts (CBC). These vials will be sent to a commercial clinical laboratory (Quest Diagnostics) for the CBC measures. The blood samples will be obtained at the same time as for the primary outcomes (prior to and following exercise as well as one, four and six hours post-exercise).

Blood will be collected in a vial containing EDTA (6 ml) for analysis of cortisol. These vials will be sent to a commercial clinical laboratory (Quest Diagnostics) for the cortisol measures. The blood samples will be obtained at the same time as for the primary outcomes (prior to and following exercise as well as one, four and six hours post-exercise).