5 and 6 chloromethyl 2 7 dichlorodihydrofluorescein diacetate acetylester cm h2dcfda

To measure cellular ROS, cells were incubated for 30 minutes in serum-free RPMI media or phosphate buffered saline (PBS) containing 5 μM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate, acetyl ester (CM-H₂DCFDA or DCF) from Sigma. The amount of ROS was quantified by a shift in green mean fluorescence intensity (MFI). To assay mitochondrial membrane potential (MMP) and superoxide in platelets, WT and KO mice were irradiated with 3 Gy TBI as previously described [4 (link)]. The irradiated mice were divided equally into two groups, one of which was given water supplemented with 250 μM MitoQ ad libitum immediately after irradiation, whereas the other group was provided with water only. Platelets were isolated 2 weeks later and incubated for 20 minutes in PBS containing 5 μM JC-1 per manufacturer’s instructions (Invitrogen). Superoxide levels were concluded with the use of Mitosox (Invitrogen) after the platelets were incubated at 37 °C for 30 minutes with 5 μM Mitosox in PBS following manufacturer’s instructions. Fluorescent intensities of the resultant cells or platelets were captured on a FACSAria and analyzed as above.