Jc 10

Mitochondrial membrane potential of cells was measured using JC-10 (Abcam). Firstly, CD19.CAR-T cells and Daudi cells were treated with either celecoxib or aspirin for 2 hours. Then JC-10 staining was performed according to the manufacturer’s instructions. For positive control, cells were stained with JC-10 working solution in the presence of 10 µmol/L carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Sigma Aldrich). Thereafter, cells were fixed by using fixation buffer (Biolegend) prior to analysis.

Mitochondria membrane potentials (MMP) were measured by JC-10 (Sigma-Aldrich, St. Louis, MI, USA) following the manufacturer’s instructions. Loss of MMP was indicated by a progressive JC-10 dislocation from mitochondria to the cytosol. Cells were photographed using fluorescent microscope (DMI8, Leica, Instruments, Germany). Red (540/570 nm) and green (485/534 nm) florescence was quantified by Leica Application Suite X (LAS X) (Leica, Milan, Italy). All the experiments were performed in triplicate. Data are expressed as the mean ± SD.

The mitochondria membrane potential (MMP) measurement is based on the fluorescence dye JC-10 and was obtained from Sigma-Aldrich (MAK159). The cells were seeded in black fluorescence 96 well plates and treated with 50 µl medium containing various supplements. After 2 h 25 µl of buffer A containing JC-10 were added. After additional 45 min 25 µl of buffer B was added. The fluorescence measurements were done at excitation 540 nm and emission 590 nm as well as excitation at 490 nm and emission at 530 nm. The ratio between the fluorescence signals excited at 540 nm/490 nm represents the MMP.

In order to evaluate mitochondrial membrane potential (ΔΨ), cells were incubated with 50 μM Rhodamine 123 (Life Technologies®), for 15 min at 4°C (protected from light), washed, and resuspended in PBS. Fluorescence intensity was measured by flow cytometry as previously described and analyzed with FlowJo software.
In a second approach to measure ΔΨ, 24 h after transfection (siRNA or overexpression), cells were incubated with the mitochondrial probe JC-10 (Sigma Aldrich). This dye forms aggregates with red fluorescence (λex = 540/λem = 590) within cells with polarized mitochondria, whereas in cells with depolarized mitochondria membrane, the dye is monomeric and green-fluorescent (λex = 490/λem = 525 nm). The ratio between red and green fluorescence was used to measure ΔΨ in cells at basal (ΔF) and uncoupling states (ΔFcccp). Briefly, cells at each experimental condition were first incubated with JC-10, and the ΔF ratio was measured. Cells were then treated with the uncoupling agent CCCP (carbonyl cyanide 3-chlorophenylhydrazone, 0.5 μM) for 15 minutes at 37°C, and the ΔFcccp was recorded. Fluorescence was quantified using the Operetta High-Content Imaging System. The ΔF/ΔFcccp ratio was calculated to obtain the relative ΔΨ at each experimental condition.

To measure mitochondrial membrane potential (ΔΨm), cells were washed with Krebs buffer (145 mM NaCl, 5 mM KCl, 10 mM HEPES, 1 mM MgCl2, 1 mM CaCl2, 5.6 mM glucose and pH 7.4/NaOH) and loaded with JC-10 (5 μM; AAT Bioquest, Inc.) at 37°C for 30 min. Green fluorescence (depolarization) was monitored at 529 nm and red fluorescence (polarized) at 590 nm on a multi-mode plate reader PHERAstar FSX (BMG Labtech). To establish that the JC-10 signal was indicative of ΔΨm, experiments were terminated inducing a maximal mitochondrial depolarization by addition of carbonyl cyanide 3-chlorophenylhydrazone (CCCP 10 μM; Sigma-Aldrich).

To detect changes in mitochondrial membrane potential (ΔΨm), cells were washed with Krebs buffer (145 mm NaCl, 5 mm KCl, 10 mm HEPES, 1 mm MgCl₂, 1 mm CaCl₂, 5.6 mm glucose and pH 7.4/NaOH) and loaded with 5 μm JC-10 (a derivative of 5,5′,6,6′-tetrachloro-1,1,3,3′-tetraethyl-benzimidazolyl-carbocyanine iodide, JC-1; AAT Bioquest, Inc.) at 37°C for 30 min. The fluorescence intensities were measured using the multi-mode plate reader PHERAstar FSX (BMG Labtech) by fluorescence excitation/emission maxima: 514/529 nm, monomer form, and 585/590 nm, aggregate form. To establish that the JC-10 signal was indicative of ΔΨm, experiments were terminated inducing a maximal mitochondrial depolarization by addition of 10 μm carbonyl cyanide 3-chlorophenylhydrazone (CCCP; Sigma-Aldrich).