Mitotracker red cmxros special packaging

Viability and cell growth were determined using the MTS assays, CellTiter 96^® AQueous One Solution Cell Proliferation Assay (Promega) and the Click-iT^® EdU Flow Cytometry Assay kit (Invitrogen) according to the manufacturer's protocol, respectively. Apoptosis was measured using annexin V - propidium iodide staining using flow cytometric analysis (Beckman Coulter, Brea, CA, USA). Study of mitochondrial membrane potential was achieved using MitoTracker Red CMXRos-Special Packaging^® (Invitrogen). Study of caspases activity was performed using staining CaspaLux^® (OncoImmunin, Gaithersburg, MD). The clonogenic activity of each BCWM1 clone was evaluated by methylcellulose culture. 500 cells were placed in 1 ml of methylcellulose medium without cytokines (MethoCult H4230, Stemcell technologies, Vancouver, Canada) in duplicates, and incubated under fully humidified conditions in 35 mm Petri dish (Greiner Bio-One, Kremsmünster, Austria) at 37°C in 5% CO2. Colonies were numbered on day 10. The following antibodies were used for immunoblotting on whole cell lysates: anti-BACH2 (LSBio^®, Seattle, WA, USA) and anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

MitoProbe JC-1 Assay Kit (M34152, Invitrogen) was used to detect mitochondrial membrane potential. Mitochondrial depolarization was detected by MitoTracker™ Green/Red (M7514, Invitrogen). Markers of healthy mitochondria were detected by MitoTracker™ Deep Red FM (M22426, Thermo). Mitochondrial ROS levels were detected by Mito Tracker Red CMXRos-special packaging (M7512, Invitrogen).