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Published on: June 4, 2019
Demonstration of the activity of P-glycoprotein by a semi-automated fluorometric method
Gabriella Spengler1, Miguel Viveiros, Marta Martins
1Unit of Mycobacteriology, UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), 1349-008 Lisboa, Portugal.
Background:
We have developed a semi-automated fluorometric method that utilizes ethidium bromide (EB), a common substrate of bacterial efflux pumps. The method is sufficiently sensitive to characterize the efflux pump systems of bacteria. Because EB is also recognized and extruded by ATP-binding cassette (ABC) transporters and these have similarity to P-glycoprotein (P-gp), the method has been extended for the evaluation of agents that can inhibit the extrusion of EB on a real-time basis by mouse lymphoma cells containing the human ABCB1 (mdr1) gene.
Materials And Methods:
Monitoring of uptake and extrusion of EB was assessed using the Rotor-Gene 3000 (Corbett Research) under different conditions.
Results:
Whereas extrusion of EB took place readily, the addition of known inhibitors of efflux pumps (verapamil, reserpine) caused retention of EB.
Conclusion:
This method is inexpensive and allows the detection of neoplastic cells with increased efflux activity as well as the screening of large numbers of compounds for inhibition of the P-gp.
