Cell culture

Human LNCaP, C4-2, and DU145 prostate cancer cells were cultured in T-medium with 10% fetal calf serum (Hyclone, Logan, UT) [8] at 37 °C in a humidified 5% CO₂ atmosphere. Upon reaching 90% confluency, cells were collected either by detachment in 0.5% trypsin containing 0.02% EDTA or detached in cold phosphate buffered saline (PBS; pH 7.41) using cell scrapers. Cells (6.5 × 10⁶) were concentrated by centrifugation at 5000 × g for 10 min, and resuspended in 1% matrigel in PBS (100 μl) for sterile injection into the flanks of athymic nude mice (vide infra). Prior studies [11] have shown that LNCaP and C4-2 cells expressed (1.05 ± 0.10) × 10⁶ and (1.67 ± 0.28) × 10⁶ PSMA molecules per cell, respectively, while DU145 cells expressed 100-fold less at only (1.35 ± 0.52) × 10⁴ PSMA molecules per cell. Subcutaneous xenografts in nude mice used LNCaP and C4-2 cells as PSMA positive samples, and DU145 cells as negative controls based on these measurements in cultured cells [6].