Irradiation leads to release of damaged exMito. exMito isolated from cell-culture supernatants of nonirradiated (CT) and irradiated (10 Gy) Caco-2 cells was subjected to (A) MTG staining and analyzed by flow cytometry. The relative MTG-positive events are shown as fold induction (n = 7). (B) Cell culture supernatants from nonirradiated (CT) and irradiated (10 Gy) Caco-2 cells cultured for 24 hours were subjected to lactate dehydrogenase (LDH) assay and the results are represented as percent (%) cytotoxicity. Staurosporine (STS; 1 μM) treatment for 12 hours was used as a positive control for the experiment (n = 3). (C) Complex-I–dependent ATP-production in 1 μg of isolated mitochondria. A representative plot of luciferase activity recorded over time is shown. (D) exMito content in the plasma of BALB/c mice 24 hours after TBI (8.8 Gy) was stained with MTG and was analyzed by flow cytometry. The number of MTG-positive events detected in 60 μL of plasma is shown; CT, nonirradiated control mice (n = 7). (E) exMito isolated from the plasma of BALB/C mice after 24 hours after TBI (8.8 Gy) was subjected to retramethylrhodamine, methyl ester (TMRM) staining in the presence or absence of complex-I substrates (pyruvate and malate) and analyzed by flow cytometry. The MFI of TMRM calculated is represented as relative fold induction (n = 4). (F) Mitochondria isolated from the plasma of BALB/C mice 24 hours after TBI (8.8 Gy) or no irradiation (CT) was subjected to complex-I–dependent ATP production (n = 3). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001; n, number of independent experiments.