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
:1. Introduction
2. Results
2.1. Characterization of S, R and T Variants of L1210 Cells
2.2. Measurement of JC-1 Fluorescence in S, R and T Cells by Fluorescence Cytometry
2.3. TQR as a Reversal Agent of P-gp-Mediated Drug Resistance in R and T Cells
2.4. Confocal Microscopy Detection JC-1 Labeling of S, R and T Cells
3. Discussion
4. Materials and Methods
4.1. Cell Cultivation Conditions and the Determination of P-gp Expression
4.2. Flow Cytometry Detection of JC-1 Fluorescence in S, R and T Cells
4.3. Confocal Microscopy of JC-1 Stained S, R and T Cells
4.4. Examination of the TQR Inhibitory Effect on P-gp Efflux Activity in S, R and T Cells using the Calcein/AM Assay
4.5. Measurement of the P-gp-Mediated Vincristine Resistance Reversal Induced by TQR
4.6. Statistical Analysis and Data Processing
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
BCRP | Breast cancer resistance protein, ABCG2 protein |
CCCP | Carbonyl cyanide 3-chlorophenylhydrazone |
CSA | Cyclosporine A |
DAPI | 4′,6-Diamidino-2-phenylindole |
ΔΨ | Mitochondrial membrane potential |
JC-1 | 5,6-Dichloro-2-[(E)-3-(5,6-dichloro-1,3-diethylbenzimidazol-3-ium-2-yl)prop-2-enylidene]-1,3-diethylbenzimidazole iodide |
MDR | Multidrug resistance |
MRP(1-3) | Multidrug resistance-associated proteins (1-3), ABCC (1-3) proteins |
MTT | ([3-(4,5-Dimethyldiazol-2-yl)-2,5 diphenyltetrazolium bromide]) |
P-gp | P-glycoprotein, ABCB1 protein |
R | P-gp-positive variant of L1210 cells obtained by selection with vincristine |
S | P-gp-negative L1210 |
T | P-gp-positive variants of L1210 cells obtained by transfection gene encoding P-gp |
TQR | Tariquidar |
VCR | Vincristine |
VER | Verapamil |
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Control | TQR (0.05 μM) | TQR (0.50 μM) | |||||
---|---|---|---|---|---|---|---|
VCR (0.25 μM) | VCR (0.25 μM) | VCR (0.25 μM) | |||||
S cells | means ± SD | 100.0 ± 14.7 | 10.5 ± 3.4 | 95.2 ± 12.8 | 11.5 ± 6.5 | 95.2 ± 10.3 | 11.8 ± 5.1 |
Student‘s t-test | t-value | 11.9 | 11.7 | 13.2 | |||
p-value | 2.1 × 10−5 | 2.4 × 10−5 | 1.2 × 10−5 | ||||
R cells | means ± SD | 100.0 ± 17.9 | 95.6 ± 9.2 | 103.4 ± 9.3 | 67.8 ± 9.5 | 95.8 ± 18.0 | 11.3 ± 5.6 |
Student‘s t-test | t-value | 0.43 | 5.4 | 9.0 | |||
p-value | 0.68 | 1.7 × 10−3 | 1.1 × 10−4 | ||||
T-cells | means±SD | 100.0 ± 17.2 | 97.2 ± 15.1 | 104.4 ± 16.6 | 36.5 ± 12.0 | 104.2 ± 15.2 | 11.5 ± 4.3 |
Student‘s t-test | t-value | 0.24 | 6.6 | 11.7 | |||
p-value | 0.82 | 5.6 × 10−4 | 2.3 × 10−5 |
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Elefantova, K.; Lakatos, B.; Kubickova, J.; Sulova, Z.; Breier, A. 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. Int. J. Mol. Sci. 2018, 19, 1985. https://doi.org/10.3390/ijms19071985
Elefantova K, Lakatos B, Kubickova J, Sulova Z, Breier A. 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. International Journal of Molecular Sciences. 2018; 19(7):1985. https://doi.org/10.3390/ijms19071985
Chicago/Turabian StyleElefantova, Katarina, Boris Lakatos, Jana Kubickova, Zdena Sulova, and Albert Breier. 2018. "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" International Journal of Molecular Sciences 19, no. 7: 1985. https://doi.org/10.3390/ijms19071985