A) Detergent treatment removes most of the CD28 found on the surface of unstimulated CD4 T cells. Resting CD4 T cells from CB6F1 mice were stained for CD3 (FITC), CD28 (PE), and CD4 (PECy5), treated with Brij-96 (Detergent) or vehicle (Controls), then analyzed by flow cytometry to evaluate CD28 fluorescence relative to CD4 fluorescence. B) TCR stimulation increases the resistance of CD28 to detergent extraction in CD4 T cells. Aliquots of untreated (left panel) or OSGE-treated (right panel) CD4 T cells were stained for CD28, stimulated by cross linking CD3, CD4, and CD28, exposed to detergent, and analyzed by flow cytometry for CD28 fluorescence. C) The histogram shows the difference between CD28 fluorescence of untreated cells (dark line) compared to OSGE-treated cells (light line). D) Quantification of CD28 associated to the detergent-resistant TCR complex. Each bar shows the relative mean CD28 retention ± SEM relative to controls (no detergent, 100%) in untreated and OSGE-treated CD4 T cells from 10 young (Y) and 10 old (O) CB6F1 mice. The asterisk (*) indicates statistical significance with respect to the young untreated group (p < 0.05).