Detection of the Mitochondrial Membrane Potential by the Cationic Dye JC-1 in L1210 Cells with Massive Overexpression of the Plasma Membrane ABCB1 Drug Transporter

Abstract

JC-1, a cationic fluorescent dye when added to living cells, is known to be localized exclusively in mitochondria, particularly in good physiological conditions characterized by sufficient mitochondrial membrane potential (ΔΨ). The accumulation of JC-1 in these organelles leads to the formation J-aggregates (with a specific red fluorescence emission maximum at 590 nm), which is in addition to the typical green fluorescence of J-monomers (emission maximum of ∼529 nm). The lack of mitochondrial ΔΨ leads to the depression of JC-1 mitochondrial accumulation and a decrease in J-aggregate formation. Therefore, the ratio between the red and green fluorescence of cells loaded with JC-1 is often used for the detection of the mitochondrial membrane potential. However, JC-1 represents a suitable substrate of the multidrug transporter P-glycoprotein (P-gp). Therefore, the depression of the JC-1 content in intracellular space and particularly in the mitochondria to a level that is inefficient for J-aggregate formation could be expected in P-gp-positive cells. In the current paper, we proved this behavior on parental P-gp-negative L1210 (S) cells and their P-gp-positive variants obtained by either selection with vincristine (R) or transfection with the human gene encoding P-gp (T). P-glycoprotein inhibitors cyclosporine A and verapamil fail to restore JC-1 loading of the R and T cells to an extent similar to that observed in S cells. In contrast, the noncompetitive high affinity P-gp inhibitor tariquidar fully restored JC-1 accumulation and the presence of the typical red fluorescence of J-aggregates. In the presence of tariquidar, measurement of the JC-1 fluorescence revealed similar levels of mitochondrial membrane potential in P-gp-negative (S) and P-gp-positive cells (R and T).

Bcl‑2 family: Novel insight into individualized therapy for ovarian cancer (Review)

“…BH3 mimetic peptide synthesized in vitro can enter into cells and successfully interact with mitochondria. JC-1, an ideal fluorescent probe, is widely used to detect mitochondrial membrane potential ( 112 ). The degree of mitochondrial apoptosis can be evaluated by detecting the color changing of the fluorescent probe.…”

Metformin as an Adjuvant to Photodynamic Therapy in Resistant Basal Cell Carcinoma Cells

“…JC-1 is a sensitive marker for mitochondrial membrane potential that forms aggregates in healthy mitochondria fluorescing in red; when the membrane potential decreases, JC-1 becomes monomers showing a green fluorescence. Therefore, the ratio of red/green fluorescence indicates the state of the mitochondrial potential [49]. JC-1 (5,5 6,6 -tetrachloro-1,1 ,3,3 -tetraethyl-benzamidazole carbo-cyanine iodide) (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) was prepared at 1 mg/mL in DMSO as a stock solution, aliquoted, and stored at −20 • C. For each analysis, cells were grown on coverslips and, 24 h after, Metf treatment cells were incubated with 1 µM JC-1 for 15 min at 37 • C. Then, cells were briefly washed with PBS, mounted on slides, and observed in situ with fluorescence using blue and green excitation light.…”

Screening of Phenanthroquinolizidine Alkaloid Derivatives for Inducing Cell Death of L1210 Leukemia Cells with Negative and Positive P-glycoprotein Expression

“…Consistent with this finding, R and T cells were much less sensitive to P-gp substrates, such as VCR, doxorubicin, and mitoxantrone, than S cells [19]. P-gp was detected by immunofluorescence confocal microscopy in R and T cells predominantly in the plasma membrane [20]. In contrast, no immunoreactive materials were visible in S cells.…”