Western Kentucky University
A Methodology for Determining Early and Late Exercise-Induced Apoptosis in Helper T (CD3+/CD4+) and Cytotoxic T Cells (CD3+/CD8+)
Institution
Western Kentucky University
Faculty Advisor/ Mentor
James Navalta
Abstract
Exercise-induced apoptosis has been assessed in lymphocytes as an entire immune cell set, typically in the early stage. Lymphocytes can be subdivided into specific subfractions (i.e. helper T, memory B cells) and apoptosis progresses through various phases (i.e. early and late stages). It's possible that exercise differentially affects subfractions, and at a variable rate of apoptosis. A methodology that allows evaluation of early and late cell death in lymphocyte subfractions is needed to understand more adequately the immune response to exercise. This study's purpose was to describe a technique for measuring early and late exercise-induced apoptosis in CD3+/CD4+ and CD3+/CD8+ cells. Male subjects who completed a treadmill test to exhaustion provided blood samples at rest and during exercise. Whole blood was added to red blood cell lysis buffer, then fixation buffer within 2-min of collection. The blood solution was incubated with antibodies specific to cell phenotype (helper T=CD3+/CD4+, cytotoxic T=CD3+/CD8+), markers of early apoptosis (annexin V+/7-AAD-), late cell death (annexin V+/7-AAD+) and necrosis (annexin V-/7-AAD+). Samples were analyzed using 4-color flow cytometry. The results for resting helper T apoptosis were 0.22±0.10% for early and 0.18±0.08% for late, and exercise-induced apoptosis was 0.38±0.18% for early and 0.10±0.1% for late. Cytotoxic T apoptosis at rest was 0.05±0.05% for early and 0.07±0.05% for late, but levels after exercise were 0.42±0.23% for early and 0.15±0.15% for late. Apoptotic measures can be obtained in lymphocyte subfractions and at varying stages of cell death.
A Methodology for Determining Early and Late Exercise-Induced Apoptosis in Helper T (CD3+/CD4+) and Cytotoxic T Cells (CD3+/CD8+)
Exercise-induced apoptosis has been assessed in lymphocytes as an entire immune cell set, typically in the early stage. Lymphocytes can be subdivided into specific subfractions (i.e. helper T, memory B cells) and apoptosis progresses through various phases (i.e. early and late stages). It's possible that exercise differentially affects subfractions, and at a variable rate of apoptosis. A methodology that allows evaluation of early and late cell death in lymphocyte subfractions is needed to understand more adequately the immune response to exercise. This study's purpose was to describe a technique for measuring early and late exercise-induced apoptosis in CD3+/CD4+ and CD3+/CD8+ cells. Male subjects who completed a treadmill test to exhaustion provided blood samples at rest and during exercise. Whole blood was added to red blood cell lysis buffer, then fixation buffer within 2-min of collection. The blood solution was incubated with antibodies specific to cell phenotype (helper T=CD3+/CD4+, cytotoxic T=CD3+/CD8+), markers of early apoptosis (annexin V+/7-AAD-), late cell death (annexin V+/7-AAD+) and necrosis (annexin V-/7-AAD+). Samples were analyzed using 4-color flow cytometry. The results for resting helper T apoptosis were 0.22±0.10% for early and 0.18±0.08% for late, and exercise-induced apoptosis was 0.38±0.18% for early and 0.10±0.1% for late. Cytotoxic T apoptosis at rest was 0.05±0.05% for early and 0.07±0.05% for late, but levels after exercise were 0.42±0.23% for early and 0.15±0.15% for late. Apoptotic measures can be obtained in lymphocyte subfractions and at varying stages of cell death.