Dcfda h2dcfda cellular ros assay kit

The live cell population was estimated using 10 μg/ml propidium iodide dye (Signa, Cat#P4170) and a BD LSRFortessa flow cytometer. Total cellular ROS was estimated using the DCFDA/H2DCFDA - Cellular ROS Assay Kit (Abcam Cat# ab113851) as per the manufacturer’s instructions. Briefly, 2 × 10⁵ cells were incubated with 20 μM of DCFDA dye for 30 minutes at 37°C. After incubation, cells were analyzed using a BD LSRFortessa flow cytometer. Mitochondria-specific reactive oxygen species (ROS) were estimated using MitoSOX Red (Molecular probes, Cat#M36008) as per the manufacturer’s instructions. Briefly, 2 × 10⁵ cells were incubated with 5 μM of MitoSOX Red dye for 10 minutes at 37°C. After incubation, cells were analyzed on a BD LSRFortessa flow cytometer.
The Mito-mKeima-based mitophagy assay was performed as per the published protocol (8 (link)). Briefly, pHAGE-mt-mKeima plasmid (a generous gift from Dr. Richard Youle) was transfected into WT or CBFB_KO MCF10A cells using Lipofectamine 3000 (Thermo Fisher, Cat# L3000001). After 24 hours, cells were harvested and analyzed using a BD FACSymphony flow cytometer. Mito-mKeima, which localizes to the matrix of mitochondria, excites at 488 nm and 561 nm at pH 4 and pH 7, respectively. Therefore, excitation shifts from 561 nm to 488 nm in lysosomes. Emission was collected at 620 nm. Data analyses were performed using FlowJo.