2,000 cells were seeded into clear-bottom 384-well plates (Nunc), allowed to attach overnight, and treated with cisplatin or vehicle the following day. After 48 hours, cells were fixed with 4% formaldehyde, blocked with 2% bovine serum albumin in Tris Buffered Saline with 0.1% TRITON X-100 (TBST), and stained with Hoechst (1:1000) and FITC-conjugated anti-γH2AX antibody (1:333, Millipore). Plates were imaged with an ImageXpress Micro automated epi-fluorescent microscope (Molecular Devices). Images were scored with MetaExpress analysis software (Molecular Devices), and statistical analysis was performed with Prism 7 (GraphPad Software). The percentage of γH2AX positive cells in cisplatin-treated samples was normalized to untreated controls.
Metaexpress analysis software
Manufactured by Molecular Devices
MetaExpress is a software solution for analyzing and managing data from high-content screening (HCS) experiments. It provides tools for image acquisition, preprocessing, and quantitative analysis of cellular and subcellular features.
Automatically generated - may contain errors
Lab products found in correlation
Clear bottom 384 well plate, by Thermo Fisher Scientific (3 mentions)
Imagexpress micro automated epi fluorescent microscope, by Molecular Devices (2 mentions)
Prism 7, by GraphPad (1 mentions)
Hoechst, by Merck Group (1 mentions)
Imagexpress micro, by Molecular Devices (1 mentions)
Fitc conjugated anti γh2ax antibody, by Merck Group (1 mentions)
Nupage kit, by Thermo Fisher Scientific (1 mentions)
3 protocols using metaexpress analysis software
1
Assessing DNA Damage in Cisplatin-Treated Cells
2
Quantitative Rad17 Immunofluorescence Assay
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Cells were seeded into clear bottom 384 well plates (Nunc), fixed with 4% formaldehyde, blocked with 2% bovine serum albumin in TBST, and stained with Hoechst and anti-RAD17 (Abnova) primary antibody followed by Alexa594 donkey anti-mouse secondary antibody. Plates were imaged with ImageXpress Micro automated epi-fluorescent microscope (Molecular Devices) and images were scored with MetaExpress analysis software (Molecular Devices). For western blot cells were lysed with RIPA buffer and prepared for SDS-PAGE using NuPAGE kit (Invitrogen). Same anti-RAD17 (Abnova) primary antibody was used as in immunofluorescence assays.
3
Quantifying DNA Damage Response
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250-500 cells were seeded into clear bottom 384 well plates (Nunc) and treated with either siRNA, kinase inhibitors, or controls. After incubation with either siRNA or small molecule inhibitor for 48-72 hours cells were fixed with 4% formaldehyde, blocked with 2% bovine serum albumin in TBST, and stained with Hoechst and FITC conjugated anti- γ-H2AX antibody (Millipore). Plates were imaged with ImageXpress Micro automated epi-fluorescent microscope (Molecular Devices) and images were scored with MetaExpress analysis software (Molecular Devices). At baseline without any pharmacological or genetic intervention, there was a non-significant trend towards more cells scoring positive for γH2AX in RAD17-KD cell lines relative to non-silencing control.
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