Rhodamine 123 efflux as a functional test for the P-gp pump. Namalwa/MDR1 cells at 5 × 105 per milliliter were treated for one hour at 37°C with 1.3 μM rhodamine 123 alone or in combination with 10 μM verapamil, 10–8 M HD37, or UIC2. Then cells were washed twice with RPMI 1640 without FBS, resuspended in FBS-free medium, and incubated for 2 more hours at 37°C to measure efflux. Namalwa cells were used as a positive control for the retention of rhodamine 123. (A) Namalwa/MDR1 cells in media (1) without rhodamine 123, (2) with rhodamine 123, and (3) with verapamil and rhodamine 123; and (4) Namalwa cells with rhodamine 123. (B) Namalwa/MDR1 cells (1) without rhodamine 123, and with rhodamine 123 in the (2) absence and (3) presence of verapamil. (4) Cells were pretreated for 10 minutes at 37°C with 10 mM MBCD prior to conducting the rhodamine 123 assay. (C) Namalwa/MDR1 cells with media (1) without rhodamine 123, (2) with rhodamine 123, (3) treated with verapamil and rhodamine 123, (4) treated with anti-CD19 (HD37) and rhodamine 123, and (5) treated with anti–P-gp (UIC2) and rhodamine 123. The histograms in each panel depict 1 representative experiment of 3 performed.
