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| | PAR Assay |
| Description: | Cellular activity of PARP-1 inhibitors was assessed by measuring the inhibition of the hydrogen peroxide induced PAR formation in HeLa cells (ECACC). Cellular PAR levels were measured by immunocytochemistry, and quantified using an ArrayScan vTi instrument (Cellomics Thermo Scientific). Studies were performed as follows: 6000 cells/well were seeded in 96 well plates (Perkin Elmer) in MEM/10% FCS and incubated for 24 hours at 37 C., 5% carbon dioxide. Test compounds were then added at the required concentration for 30 minutes. DNA damage was then induced adding hydrogen peroxide at the concentration of 0.1 mM for 15 minutes. Concentration curves were prepared in MEM/10% FCS from compound stocks in DMSO, and final DMSO concentration was 0.002% (v/v). Duplicate wells for each concentration point were prepared with a typical highest compound concentration of 20 uM and serial dilution 1:3. Plates were dried and fixed adding cold methanol-acetone (70:30) solution for 15 minutes at room temperature. |
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