IEC and EC necroptosis are triggers of TNF-induced death of Ripk1 ^S161E/S161E mice. (A) Flow cytometry analysis of counts of total immune cells and different subsets in the peripheral blood of 8- to 12-wk-old male littermate Ripk1^+/+ and Ripk1^S161E/S161E mice after TNF injection (50 μg/kg, i.v.) at the indicated time points. (B) Immune cell compositions of the thymus (Thy), spleen (SP), lymph node (LN), and bone marrow (BM) of untreated Ripk1^+/+ and Ripk1^S161E/S161E mice were analyzed using flow cytometry. All mice were 8- to 12-wk-old male littermates. The number of mice used was as indicated. (C) Cecal IECs and ECs were isolated from male Ripk3^f/fVillin-cre^+/− mice, Ripk3^f/fTie2-cre^+/− mice, and their littermate Ripk3^f/f mice, and tamoxifen-treated male Ripk3^f/fCdh5-ERT2-cre^+/− mice and their littermate Ripk3^f/f mice, respectively. RIPK3 expression in the cell lysates was examined by immunoblotting. E-cadherin and endomucin were used as markers for IECs and ECs, respectively. Each number represents one mouse. The above experiments were independently performed twice. (D) Representative immunofluorescence staining images of ceca from five mice of each indicated genotype after TNF treatment for 6 h (50 μg/kg, i.v.). All mice were 8- to 12-wk-old male littermates. E-Cad (red), cleaved caspase-3 D175 (CC3, green). Scale bars, 50 μm. Percentages of CC3⁺ cells in E-Cad⁺ cells were quantified using ImageJ. Data are presented as mean ± SD. P values are determined by a two-tailed, unpaired t test. ns, P ≥ 0.05. (E and F) 8- to 12-wk-old littermate male WT mice (A) and Ripk3^−/− mice (B) were injected with PBS or TNF (400 μg/kg, i.v.) for 4 h and then EB (0.5 mg/mouse) for 20 min. Organs were collected, and the amount of EB extracted from the organs was measured by spectrophotometry. Data are presented as mean ± SD. P values are determined by a two-tailed, unpaired t test. **P < 0.01. Source data are available for this figure: SourceData FS4.