Uramustine

Cell experiment:

The K562 human chronic myeloid leukemia cells are maintained in RPM1 1640 medium supplemented with 10% fetal calf serum and 2 mM glutamine at 37°C in a humidified atmosphere containing 5% CO2 and are incubated with a specified dose of drug for 1 h at 37°C in the dark. The incubation is terminated by centrifugation (5 min, 300 g), and the cells are washed once with drug-free medium. Following the appropriate Uramustine treatment, the cells are transferred to 96-well microtiter plates, with 104 cells per well and 8 wells per sample. Plates are then kept in the dark at 3°C in a humidified atmosphere containing 5% CO2. The assay is based on the ability of viable cells to reduce a yellow soluble tetrazolium salt, MTT to an insoluble purple formazan precipitate. Following incubation of the plates for 4 days (to allow control cells to increase in number by 10-fold), 20 μL of a 5 mg/mL solution of MTT in phosphate-buffered saline is added to each well and the plates are further incubated for 5 h. The plates are then centrifuged for 5 min at 300 g, and the bulk of the medium is pipetted from the cell pellet, leaving 10?20 μL per well. A total of 200 μL of DMSO is added to each well, and the samples are agitated to ensure complete mixing. The optical density is then read at a wavelength of 550 nm on a plate reader, and the dose?response curve is constructed. For each curve, an IC50 value is read as the dose required to reduce the final optical density to 50% of the control value[1].

References:

[1]. Baraldi PG, et al. Design, synthesis, and biological activity of hybrid compounds between uramustine and DNA minor groove binder distamycin A. J Med Chem. 2002 Aug 15;45(17):3630-3638.