Mitoprobe jc 1 assay kit

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).

To observe the changes in the mitochondrial membrane potential resulting from the combination treatment of 2-DG and plasma, JC-1 staining was performed on treated blood cancer cells using the MitoProbe JC-1 assay kit (Invitrogen, USA) as previously described59 (link)60 . Caspase activities were measured using the Caspase-Glo® 3/7 assay kit, and Caspase-Glo® 9 assay kit (Promega) on a white 96-well flat-bottom microplate according to the manufacturer's instructions. To verify caspase-dependent cell death, we pre-incubated the cells with Z–VAD FMK, a pan-caspase inhibitor, for a viability analysis before 2 h of combination treatment. Next, to further verify apoptosis, the treated cells were subjected to a FACS analysis. Briefly, at 24 h, cells were harvested and stained with annexin V-FITC/PI using the Annexin V: FITC Apoptosis Detection Kit I (BD Biosciences, Seoul, Korea) and directly analyzed using a flow cytometer (BD FACSVerse, NJ, USA) and the FACS suite software.

MMP was assessed using the Mito Probe JC-1 assay kit (Invitrogen). Cells were incubated with 2 μM JC-1 for 15–30 min. The red and green signals of JC-1 were visualized using a fluorescence microscope. The ratio of red/green fluorescence intensity was calculated as a measure of MMP.

To detect changes in the mitochondrial membrane potential after treatments, JC-1 staining was performed on treated cells with MitoProbe JC-1 assay kit (Invitrogen, Carlsbad, CA, USA). JC-1 is a sensitive marker for the detection of the mitochondrial membrane potential in apoptotic cells. JC-1 shows green fluorescence (due to the depolarized mitochondrial membrane) inside the cell. However, normal polarized mitochondrial membrane cells show red fluorescence. For positive MMP depolarization, we used cccp, which abolishes the membrane potential quickly. The protophore cccp mainly induces mitochondrial permeability transition in cancer cells. In our experiment, we compare our results with cccp as control. Therefore it may be possible that metalla cycles can induce transient nature of mitochondrial membrane potential change in cancer cells [58 (link)].

To assess MMP, staining with cationic dye JC-1 was performed. Cells were seeded at a density of 1 × 10⁶ cells mL⁻¹ and treated with or without 30 mM 2-deoxy-D-ribose. After 24 h cells were collected, centrifuged (400×g, 4 °C, 5 min) and washed twice with PBS. After washing, cells were incubated for 20 min in the incubator (37 °C, 5%CO₂) with 10 μl JC-1 staining solution (MitoProbe Jc-1 Assay Kit, Invitrogen) diluted in 1 ml PBS. Healthy cells with functional mitochondria containing red JC-1 aggregates are detectable in FL-2 channel. Apoptotic or unhealthy cells with collapsed mitochondria contain mainly green JC-1 monomers and are detectable in FL-1 channel. As a positive control we used carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which is a chemical inhibitor of oxidative phosphorylation and causes an uncoupling of the proton gradient. Measurements were performed using FACSCalibur instrument (Becton Dickinson). Analysis was made with BD Cell Quest Pro Software.

The 3D cultured organoids were isolated as a single cell separately by trypsinization, washed three times with PBS, and made into a cell suspension. The MitoProbe™ JC-1 Assay Kit (Invitrogen, M34152) was used to detect mitochondrial membrane potential. The cell suspension was incubated for 20 min with 10 ug/ml JC-1 at 37°C in the dark. The cells were incubated for 15 min with the FITC-Annexin V/Dead Cell Apoptosis Kit (ThermoFisher company, V13242) in the dark at room temperature for early apoptosis and immediately analyzed by flow cytometry according to the instructions. The TUNEL assay was used to detect late apoptosis, and the cells were incubated only with PI for the cell cycle analysis according to the instructions.