3h docetaxel

Cellular drug accumulation was determined using a published method (6 (link)). Briefly, 1 × 10⁶ cells were seeded in six-well dishes and allowed to attach overnight. [³H]-docetaxel (10 nmol/L, American Radiolabeled Chemicals, St. Louis, MO) was allowed to accumulate for 1 hr at 37 °C with and without 2 μmol/L PSC-833, aspirated, and dishes were washed once with ice-cold PBS. Cells were lysed immediately using a 2% (w/v) SDS solution, and counts were determined upon the addition of EcoLite liquid scintillation cocktail (MP Biomedicals, Solon, OH), and normalized to protein content.
These data were confirmed by determining the accumulation of rhodamine-123 and BODIPY-paclitaxel (both Life Technologies) by flow cytometry. Cells were harvested, exposed to either rhodamine-123 or BODIPY-paclitaxel for 1 hr at 37 °C, drug was removed by centrifugation (200 × g) at 4 °C, and cells washed once in cold PBS. In order to correlate drug accumulation with P-gp content, cells were stained on ice using an anti-P-gp mouse monoclonal which detects an external epitope (clone UIC2, EMD Millipore, Billerica, Ma), and detected by a Texas-Red goat anti-mouse secondary antibody (Life Technologies) using an LSR II flow cytometer (BD Biosciences, San Jose, CA). The effects of 2 μmol/L PSC-833 and other known MDR modulators were accessed in separate experimental conditions.