HeLa WT and cavin3 KO cells were counted using a hemocytometer and seeded into 96-well plate at 1000 cells/well (eight wells for each treatment) in 90 ml medium per well. Cells were either left untreated or were treated with 90% hypo-osmotic media (90% water in DMEM), UV treatment for 2 min without media with a UV germicidal light source (UV-C 254 nm) or 200 μM H2O2. After stress addition, 10 ml of PrestoBlue Viability Reagent (10×) (Absorbance wavelength: 600 nm) (Thermo Fisher Scientific) was added to cells. The PrestoBlue reagent was incubated constantly in wells over a time course from 2 hr to 24 hr. Control wells containing only cell culture media (no cells) were included in triplicate on each plate for background fluorescence calculations. Plates were returned to a 37°C incubator. Both absorbance values at 570 nm and 600 nm were measured for each plate in a TECAN Infinite 200 Pro reader (Millennium Science), where 570 nm was used as the experimental wavelength and 600 nm as normalization wavelength.

For PARP inhibitor experiments, cells were either left untreated or were treated with PARP inhibitor (AZD2461 5 nM) for 6 days after which 10 μl of PrestoBlue Viability Reagent (10×) (absorbance wavelength: 600 nm) (Thermo Fisher Scientific) was added to cells. Control wells containing only cell culture media (no cells) were included in triplicate on each plate for background fluorescence calculations. Plates were returned to a 37°C incubator. Both absorbance values at 570 nm and 600 nm were measured for each plate in a TECAN Infinite 200 Pro reader, where 570 nm was used as the experimental wavelength and 600 nm as normalization wavelength.

Raw data was processed to evaluate the percent reduction of PrestoBlue reagent for each well by using the following equation referring to the manufacturer’s protocol:

where A1 is the absorbance of test wells at 570 nm, A2 is the absorbance of test wells at 600 nm, N1 is the absorbance of media-only wells at 570 nm, and N2 is the absorbance of media-only wells at 600 nm.

注意:以上内容是从某篇研究文章中自动提取的,可能无法正确显示。



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