Measuring Intracellular Drug Accumulation with [3H]-Saquinavir

Radio-labeled ³H-SQV (specific activity: 1.0 Ci/mM) was obtained from Moravek Biochemicals (Brea, CA, USA) and was used to measure intracellular drug accumulation [7 (link)]. Briefly, HuT78 cells were cultured in 24-well plates (5 × 10⁵ per well) and pre-exposed to CyO2 (0.5 and 1.5 μM) for either 10 min or 30 min, followed by washing off with PBS and addition of ³H-SQV (1.7 pM) and incubation at 37 °C for 2 h. Cells were harvested by centrifugation and extracts obtained by lysing with 1.0 M ammonium hydroxide (NH₄OH). Intracellular levels of ³H-SQV were monitored in the lysates, and 100 μL of the lysate was used to measure protein levels by using the BCA protein assay kit (ThermoFisher, Waltham, MA, USA). The remaining 100 μL of the lysate was dissolved in 10 mL of EcoLite scintillation fluid from MP Biomedicals (Santa Ana, CA, USA) and count per minute (CPM) were determined by using a Tri-Carb 2800TR Liquid Scintillation counter (Perkin Elmer, Waltham, MA, USA). In respective samples, data were normalized to the protein contents and presented as CPM/μg of protein.