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. 1998 Oct 19;188(8):1391–1399. doi: 10.1084/jem.188.8.1391

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Surface-bound TNF-α is dependent upon antigen density and is not correlated with apoptosis. 5 d after stimulation, high avidity CTLs were stimulated with Thy 1.2–depleted spleen cells pulsed with various concentrations of I10 peptide. After 24 h in culture, cells were harvested and washed, and surface-bound TNF-α was detected by FITC-conjugated antibody using flow cytometry, gating only Thy 1.2–positive cells to exclude antigen presenting cells. The mean fluorescence was determined for CTLs in both the viable and apoptotic gates based on forward versus side scatter. Apoptosis was measured in the same samples as the number of CTLs in the apoptotic gate taken as a percentage of the total CTLs in both viable and apoptotic gates. Surface-bound TNF-α increased at optimal levels of peptide, two logs lower than peptide concentrations sufficient to cause apoptosis under these conditions. Similar results were obtained in three additional experiments.