(2010) and Spielmann et?al

(2010) and Spielmann et?al. Pro software (BD Biosciences, USA) on a BD FACS Calibur four\color flow cytometer equipped with a 15 mW argon ion laser emitting light at fixed wavelength of 488?nm (BD Biosciences, USA). First, lymphocyte population was gated using forward scatter and side scatter. CD3+ events were gated, followed by gating of CD4+ and CD8+ populations. Subsequent expression of CD31 was gated for, and these cells were assessed for expression of CD28. Representative flow cytometry dot plots is provided in Figure?1; 10,000 lymphocytic events were measured per sample. Circulating concentrations of T cells and subsequent subsets were obtained using a dual platform method, by multiplying the percentage values obtained from the flow cytometer by the corresponding lymphocyte counts as obtained from hematology analysis. Open in a separate window Figure 1 Flow cytometric quantification of CD31+ CD28+/null TANG cells. Side scatter vs. forward scatter for identification of lymphocyte gate (A), CD3+ gating for identification of T cells (B), identification of CD4+ (C) or CD8+ (D) T cells followed by identification of CD31+ and CD31?subsets (E). CD31+ subsets were then analyzed for expression of CD28 (F). Histogram data shows isotype control (black lines) and sample (red lines). Changes in blood volume were accounted for by using known measures of hematocrit and hemoglobin obtained from automated hematology analysis (Sysmex, XS 1000i, UK) (Dill and Costill 1974). Statistical analysis All data are presented as mean??SEM unless otherwise stated. Independent = 11.583, = 22.107; = 3.731; = 13.718; = 10.313; = 5.250; = 11.583; = 3.198; = 2.153; = 6.384;= 0.000;= 0.139;= 2.834;= 1.098;= 2.375, P?=?0.045) of CD28null CD8+ TANG cells than CD28+ CD8+ TANG cells (Fig.?4). Open in a Sulfacarbamide separate window Figure 4 Exercise responsiveness of CD28+ and senescent\associated CD28null TANG cells in young (n?=?9; A and C) and older (n?=?10; B and D) men. *Significant main effect of exercise, ??significant exercise phenotype interaction effects (P?P?P?=?0.098 for trend), despite >280?cellsL?1 difference between young and older men in our study Sulfacarbamide (total TANG cells), which may be of clinical significance. Interestingly, we also show that in the young population (18C25?years) that there were no differences Sulfacarbamide in the response of CD28null and CD28+ TANG cells; however, in the older population (60C75?years), there was a greater responsiveness of CD28null than CD28\expressing CD8+ TANG cells. Our lab has previously shown that exercise significantly increases the number of circulating TANG cells (Ross et?al. 2016), and older adults display reduced resting and exercise\induced mobilization of TANG cells into the circulation in response to an exercise Rabbit Polyclonal to CCNB1IP1 bout (Ross et?al. 2018). Reductions in basal TANG cells in older adults may be due to thymic involution (Simpson 2011); however, we do observe an increase in CD28null TANG cells in the older population. CD28 expression is lost on repeated rounds of T\cell division.