Cell Surface Labeling and 'TUNEL'
As circulating peripheral lymphocytes are selected for lineage commitment within the the mouse thymus the arising T cell population goes through very defined stages of development. Initially double negative CD4-/8- T cells which comprise 1-2% of the T cell ontogeny have greater life span conferred from an upregulation of the anti-apoptosis oncogene Bcl-2 and therefore less programmed cell death. Another stage of lineage development is to progress to the double positive CD4+/8+ compartment which typically represents >80% of the T cell population. Due to the down regulation of Bcl-2 there is an increase in apoptosis within this DP population. The final stage is full lineage commitment into single positives CD4+/8- (8-12%) and CD4-/8+ (2-4%) where the lowest expression of Bcl-2 and greatest amount of apoptosis is present.
Below is an example of the gating and quantitation of apoptosis in two T cell subsets. The first dot plot is the morphology of cultured Mouse thymus cells. Two populations are seen. The live gate is used to generate the second dot plot (CD4-PE/8-FITC profile) as well as the two histograms (dUTP-APC). Decreased cell volume and increased cellular side scatter, characteristics of advanced apoptosis are shown in the gate just left of the live gate.
Demonstration of Gating and Quantitation of Apoptosis in T Cell Subsets
Example from Research Core 3: Molecular Genetics and Environmental Carcinogenesis. Ellen Richie, Ph.D. & Heather Poetschke Klug, Ph.D.
Cell Surface Labeling and 'TUNEL' Protocol
