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.
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 humanABCB1 (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.