LPS-induced caspaselike activation is specific, rapid, and sustained in time. (A) Untreated (○), zVAD (40 μM) treated (•), LPS (100 ng/mL) stimulated (▵), or zVAD pretreated (1 hour) and LPS-stimulated (100 ng/mL) (▴) DCs were analyzed for PI uptake by flow cytometry at the indicated time points. Means ± 1 SD from 5 different experiments are shown. (B) Untreated (○), zVAD pretreated (•), IL1 (100 ng/mL) stimulated (▵), or zVAD pretreated and IL1-stimulated (▴) DCs were analyzed for PI uptake by flow cytometry at the indicated time points. Means ± 1 SD from 3 different experiments are shown. (C) DCs were pretreated with different doses of zVAD before LPS stimulation. Cells were analyzed after 8 hours for PI uptake by flow cytometry. (D) DCs were pretreated with 40 μM zVAD and then stimulated with different doses of LPS. After 8 hours cells were analyzed for PI uptake by flow cytometry. (E) DCs were pretreated with YVAD (up to 160 μM), IETD (up to 40 μM), zFA (up to 160 μM), WEHD (up to 20 μM), or zVAD (40 μM) and exposed to LPS. Cell death was analyzed by FITC–annexin V staining and flow cytometry after 24 hours. Means ± 1 SD from 4 different experiments are shown. (F) DCs were exposed to LPS. At the indicated time points, cells were washed 3 times and put back in colture with or without zVAD. After 12 hours cells were analyzed for PI uptake by flow cytometry. Means ± 1 SD from 3 different experiments are shown. (G) DCs left without additional treatment (○) or treated with zVAD (•) at different time points after LPS stimulation. Eight hours after zVAD addition, cells were analyzed for PI uptake by flow cytometry. Means ± 1 SD from 3 different experiments are shown. (H) A typical experiment was performed in parallel at 37°C and at 4°C. Twenty-four hours after LPS exposure zVAD pretreated DCs were analyzed for PI uptake by flow cytometry. Means ± 1 SD from 3 different experiments are shown.