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Flow cytometric analysis of P-glycoprotein function using rhodamine 123
1Departament de Criobiologia i Terapia Cellular, Institut de Recerca Oncologica, Hospital Duran i Reynals, Barcelona, Spain.
Leukemia
|July 1, 1997
Summary
This study optimizes Rhodamine 123 measurements to assess P-glycoprotein (P-gp) efflux activity in leukemia cells, improving cancer drug resistance phenotyping. Findings guide better clinical strategies for overcoming multidrug resistance (MDR).
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- P-glycoprotein (P-gp), encoded by the MDR1 gene, is a transmembrane efflux pump contributing to cancer treatment failure.
- Rhodamine 123 (Rh123) is a fluorescent probe used to measure P-gp efflux activity and assess drug resistance phenotypes.
- Accurate measurement of P-gp activity is crucial for understanding and overcoming multidrug resistance (MDR) in cancer.
Purpose of the Study:
- To optimize the use of Rhodamine 123 (Rh123) for measuring P-glycoprotein (P-gp) functional activity in human leukemic cell lines.
- To identify critical variables affecting Rh123 efflux measurements, including Rh123 concentration, cell viability, and culture conditions.
- To evaluate the efficacy of P-gp inhibitors and the influence of fetal calf serum on chemosensitizer bioavailability.
Main Methods:
- Flow cytometry was employed to analyze Rh123 influx and efflux rates in human leukemic cell lines.
- Life-gating techniques were utilized to ensure accurate measurements of Rh123 efflux.
- Various concentrations of Rh123 and different culture conditions, including the presence or absence of fetal calf serum, were tested.
- The relative efficiency of P-gp inhibitors (PSC-833, cyclosporin A, verapamil) was assessed.
Main Results:
- Optimal non-cytotoxic concentrations of Rh123 for accurate measurements were determined to be 50-200 ng/ml.
- Life-gating enabled precise measurement of the average Rh123 efflux rate.
- The relative efficiency of P-gp inhibitors followed the order: PSC-833 > cyclosporin A > verapamil.
- Fetal calf serum generally did not affect chemosensitizer bioavailability, except in serum-free conditions where PSC-833 showed significantly reduced P-gp activity.
Conclusions:
- The optimized experimental strategy using Rh123 and flow cytometry provides a reliable method for studying cellular drug resistance phenotypes.
- Accurate phenotyping of P-gp activity can guide clinical decisions in cancer treatment.
- Understanding the impact of culture conditions and P-gp modulators is essential for effective chemosensitization strategies.