Matrix 96 β counter

The prostate cancer cell lines PC3 and DU145 were washed in HBSS four times before seeded into 96-well culture plates with 10 % FCS in RPMI or DMEM, respectively. Next, the cells were treated with various concentrations of the PRL-3 inhibitor I or DMSO. After 56 h, cells were pulsed with 1 µCi of methyl-[3H]-thymidine (NEN Life Science Products, Boston, MA, USA) per well and harvested 16 h later with a Micromate 96-well harvester (Packard, Meriden, CT, USA). The radiation was then measured with a Matrix 96 β counter (Packard).

Ten microliter of cell culture supernatant was collected into a 96-well plate every other day and subjected to RT activity assay as previously described [68 (link)]. Briefly, 10 μl of transfected and/or infected supernatant was incubated at 37 °C overnight in the presence of 25 μl RT mixture (1 μl of fresh 10 mCi/mL [α-32P]-dTTP in 1 ml RT master mix: 50 mM Tris-HCl (pH 7.8), 75 mM KCl, 2 mM DTT, 5 mM MgCl₂, 5 μg/ml of poly(rA), 6.25 μg/ml oligo(dT), and 0.5 % (v/v) NP40). After incubation, 10 μl of the reaction mixture was blotted from each well onto the 96-well format DEAE filtermat. After 10 min of drying, the filtermat was washed five times with 1 × SSC solution and twice with 85 % ethanol (5 min for each time) in a shaking platform. The filters were then wrapped with Saran wrap and either exposed overnight onto autoradiography film (Kodak BioMax MR) or counted with a Matrix 96 β-counter (Packard, Meriden, CT) to acquire the quantitative RT activity.