Image it live mitochondria permeability transition pore assay kit

Calcein fluorescence was measured using the Image-IT live mitochondria permeability transition pore assay kit (Thermo Fisher Sci.) as per the manufacturer’s protocol. Briefly, cells seeded on a four-chamber cover glass (Lab-Tek) were loaded with calcein AM (1 µM), MitoTracker Red (200 nM), treated with or without CoCl₂ (1 mM) in Hank’s balanced salt solution (HBSS), and incubated for 15 min at 37 °C under 5% CO₂. Cells were then washed with HBSS buffer and images were taken under an Olympus 1 × 71 fluorescence microscope using a ×10 objective with or without the ×1.6 magnification changer.
For flow cytometry, cells (1 × 10⁴) were harvested using Trypsin-EDTA and pre-incubated with CsA (1 µM), SFA (2 µM), XeC (3 µM) or RuR (3 µM) for 1 h then stained as indicated above. Cells were then washed with HBSS, resuspended in ice-cold PBS and analyzed by flow cytometry using a fluorescein isothiocyanate filter (530 nm) for measuring calcein fluorescence.

RPMI 1640 media, fetal bovine serum, penicillin-streptomycin, Mag-Fluo-4 AM, Rhod-2 AM, Fluo-4 AM, Annexin V-FITC staining kit, Image-IT live mitochondria permeability transition pore assay kit, MitoSOX Red, MitoTracker green and DCFDA were from Thermo Fisher Scientific (Burlington, ON, Canada). L-asparaginase (ab73439) was from Abcam (Toronto, ON, Canada). 2,5-di-tert-butylhydroquinone (TBHQ) was from Sigma (Oakville, ON, Canada). Xestospongin-C (XeC), ruthenium red (RuR), and cyclosporine A (CsA) were from Bio-Techne (Oakville, ON, Canada). HAP1 (D-12) and actin (I19) antibodies, and Mito-Tempo were from Santa Cruz Biotech. (Dallas, TX, United States of America).