Clonogenic Potential of LNCaP Cells Exposed to 225Ac-Radioconjugates

Colony formation of LNCaP cells upon exposure to increasing activity concentrations of both ²²⁵Ac-radioconjugates was analyzed as previously reported 25 (link) with slight modifications. Briefly, 6000 LNCaP cells were seeded in 6-well microplates (Greiner Bio-One GmbH, Frickenhausen, Germany) and cultivated overnight to allow cell adhesion. The cell culture medium was replaced by serum-free RPMI-1640 medium (2 mL/well) and the microplates were further incubated at 37 °C. After 1 h, five different activity concentrations (0.05, 0.5, 1, 5, and 50 kBq/mL) of the ²²⁵Ac-radioconjugates were added in triplicate. After 1 or 4 h of incubation at 37 °C, the supernatants were replaced by fresh RPMI-1640 medium with 10% FCS (2 mL/well) and plates were incubated at 37 °C for 8 d. Finally, the cell culture medium was discarded and colonies were stained with 0.5% crystal violet in 50% methanol (1 mL/well) for 30 min, after which the plates were rinsed three times with deionized water and subsequently air-dried. The plates were scanned with an Amersham Typhoon 5 Scanner (Cytiva Europe GmbH, Freiburg, Germany) and the colonies were counted using the Image-Quant TL software (Version 8.1, Cytiva Europe GmbH, Freiburg, Germany).