Submitted to: Journal of Federation of American Societies for Experimental Biology
Publication Type: Abstract Only
Publication Acceptance Date: 11/7/2006
Publication Date: 4/1/2007
Citation: Idso, J.P., Hunt, C., Watts, J.J., Combs, G.F. 2007. New methodology for quantification of human white blood cell (WBC) apoptosis following exposure of whole blood samples to cyclohexamide (CHX) or hydrogen peroxide (HP) as assessed by flow cytometry [abstract]. Journal of Federation of American Societies for Experimental Biology. 21(6):A758.
Technical Abstract: The ability of WBC to undergo apoptosis is an essential feature in the maintenance and regulation of the immune response. We have developed a method for the assessment of WBC potential for apoptosis (high expression of 3´-hydroxyl ends in DNA) in a whole blood in vitro system. Whole blood collected from free-living individuals (n = 30) was held (15 - 60 min; room temp; heparinized glass vacutainers) then aliquoted for incubation (180 min at 37o C) with the apoptosis inducers, HP (25.4 mmol) or CHX (9.5 mmol), or with no inducer for 0 min or 180 min. Following lysing of RBC, washed WBC were fixed (1% paraformaldehyde; 60 min) and permeabilized (70% ETOH; 18 h). Flow cytometry differentiated WBC by size and complexity, apoptotic subpopulation (fluorescein labeled antiBrdU monoclonal antibody) and cellular DNA content (propidium iodide). Bivariate analysis and logical gating techniques based on the distribution of a consistently identifiable cell population allowed for the standardized identification of apoptotic cells. An apoptotic control standard (human lymphoma cell line) confirmed the labeling reagents performance. Mean percent change in apoptosis was significantly greater in HP (3.95 %), and CHX (5.36 %), treatments, than at 0 min (1.04 %, p < 0.0003), but not at 180 min (1.95 %, p = 0.21). This method appears capable of measuring the apoptotic potential of WBC maintained in a near physiologic environment.