BJ fibroblasts, MEF, iPSC or dFib-indOSK fibroblast-like cells were plated on μCLEAR bottom 96-well plates (Greiner Bio-One). One day later, cells were untreated, infected by spinfection twice and/or incubated with nucleosides or doxycline as indicated. γH2AX staining was performed using standard procedures as described24 (link) and images were automatically acquired in an Opera High-Content Screening System (Perkin Elmer). Each condition was performed in triplicate and at least 25 different fields per well at a × 20 magnification were taken. Images were segmented using a DAPI (4′,6-diamidino-2-phenylindole) staining to generate nuclear masks from which the mean γH2AX signal was calculated.
μclear bottom 96 well plate
Manufactured by Greiner
The μCLEAR bottom 96-well plates are a type of laboratory equipment designed for use in various scientific applications. They feature a clear bottom that allows for improved imaging and detection of samples within the wells.
Automatically generated - may contain errors
Lab products found in correlation
Opera high content screening system, by PerkinElmer (6 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Poly l lysine coated, by Merck Group (2 mentions)
Alexa fluor 488 from molecular probes, by Thermo Fisher Scientific (2 mentions)
Mouse monoclonal anti phospho histone h2ax antibody, by Merck Group (2 mentions)
Prism software, by GraphPad (1 mentions)
Click it edu alexa fluor imaging kit, by Thermo Fisher Scientific (1 mentions)
Imagexpress pico automated cell imaging system, by Molecular Devices (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Columbus software, by PerkinElmer (1 mentions)
7 protocols using μclear bottom 96 well plate
1
Quantifying DNA Damage via γH2AX
2
High-Throughput Immunofluorescence Imaging of Chromatin-Bound Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunofluorescence of chromatin bound proteins, cells were seeded on 0.1% gelatin, then the soluble material was pre-extracted with CSKI buffer for 4-6 minutes (10 mM Pipes, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, and 0.5% Triton X-100) before fixation in mSTF buffer (150 mM 2-Bromo-2-nitro-1,3-propanediol, 108 mM diazolidinyl urea, 10 mM Na Citrate, 50 mM EDTA (pH 5.7)). Cells were permeabilized in 100 mM Tris-HCl (pH 7.4), 50 mM EDTA (pH 8.0), 0.5% Trion X-100 followed by the staining for specific proteins using standard protocols.
For high throughput microscopy, cells were grown on μCLEAR bottom 96-well plates (Greiner Bio-One) and immunofluorescence was performed using standard procedures. Analysis of DNA Replication by EdU incorporation was done using Click-It (Invitrogen, Thermo Fisher Scientific) following manufacturers’ instructions.
In all cases, images were automatically acquired from each well using an Opera High-Content Screening System (Perkin Elmer). A 20x magnification lens was used and images were taken at non-saturating conditions. Images were segmented using DAPI signals to generate masks matching cell nuclei from which the mean signals for the rest of the stainings were calculated. Data were represented with the use of the Prism software (GraphPad Software).
For high throughput microscopy, cells were grown on μCLEAR bottom 96-well plates (Greiner Bio-One) and immunofluorescence was performed using standard procedures. Analysis of DNA Replication by EdU incorporation was done using Click-It (Invitrogen, Thermo Fisher Scientific) following manufacturers’ instructions.
In all cases, images were automatically acquired from each well using an Opera High-Content Screening System (Perkin Elmer). A 20x magnification lens was used and images were taken at non-saturating conditions. Images were segmented using DAPI signals to generate masks matching cell nuclei from which the mean signals for the rest of the stainings were calculated. Data were represented with the use of the Prism software (GraphPad Software).
3
High-Throughput DNA Damage Response Analysis
4
Clonogenic Survival and Cell Viability Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For clonogenic survival assays, 2,000 cells were plated in six‐well tissue culture plates in the corresponding culture medium. The following day, cells were treated with the indicated concentrations of drugs. Cells were maintained with the compounds for 10 days, changing the medium every 2–3 days, and then fixed and stained with 0.4% methylene blue in methanol for 30 min. Clonogenic assays were quantified following a previously described pipeline (Guzman et al, 2014 (link)). Cell viability was also measured by High‐Throughput Microscopy. In brief, 3,000 cells were seeded per well in μCLEAR bottom 96‐well plates (Greiner Bio‐One) and treated with the indicated concentrations of drugs the following day. Seventy‐two hours later, cells were fixed with 4% PFA and permeabilised with 0.5% Triton X‐100, following standard procedures. Cells were subsequently stained with DAPI and images were automatically acquired from each well using an Opera High‐Content Screening System (Perkin Elmer) or an ImageXpress Pico Automated Cell Imaging System (Molecular Devices). 20× or 10× magnification lenses were used indifferently, and images were taken at nonsaturating conditions. Images were then segmented using DAPI signals to generate masks that allowed the quantification of nuclei per condition.
5
High-throughput imaging of pluripotent stem cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following procedures previously described in (47 ), a total of 10,000 to 20,000 ESC were plated under 2i/LIF conditions on gelatin or fibronectin (5 μg/ml; Millipore) in μCLEAR bottom 96-well plates (Greiner Bio-One, 655087). ESC were either maintained under 2i/LIF conditions or treated with FGF2/Activin-A for the indicated times. Staining was performed using standard procedures. DNA was stained using DAPI.
Images were automatically acquired using a CellVoyager CV7000 high-throughput spinning disk confocal microscope (Yokogawa, Japan). Each condition was performed in triplicate wells, and/or at least nine different fields of view were acquired per well. High content image analysis was performed using the Columbus software (PerkinElmer). In brief, nuclei were first segmented using the DAPI channel. Mean fluorescence intensities for OTX2, ERF, ESRRB, or NANOG signal were calculated over the nuclear masks in their respective channels. Single-cell data obtained from the Columbus software were exported as flat tabular .txt files, then analyzed using RStudio version 1.2.5001, and plotted using GraphPad Prism version 9.0.0 (47 ).
Images were automatically acquired using a CellVoyager CV7000 high-throughput spinning disk confocal microscope (Yokogawa, Japan). Each condition was performed in triplicate wells, and/or at least nine different fields of view were acquired per well. High content image analysis was performed using the Columbus software (PerkinElmer). In brief, nuclei were first segmented using the DAPI channel. Mean fluorescence intensities for OTX2, ERF, ESRRB, or NANOG signal were calculated over the nuclear masks in their respective channels. Single-cell data obtained from the Columbus software were exported as flat tabular .txt files, then analyzed using RStudio version 1.2.5001, and plotted using GraphPad Prism version 9.0.0 (47 ).
6
Quantification of DNA Damage Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LCLs were cultured 4 hours before fixation with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, Philadelphia, USA). Two hours before fixation, cells were counted and seeded into a poly-L-lysine-coated (Sigma-Aldrich) μCLEAR bottom 96-well plate (Greiner Bio-One) at a density of 75,000 cells per 100ul full media per well. LCL were then left for 2 hours in order to attach to the surface of the wells, fixed for 15 min at room temperature, permeabilized in 0.5% Triton X-100 in PBS for 20 minutes at 4°C and stained with primary and secondary antibodies and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) to visualize nuclei. To detect γ-H2AX we used mouse monoclonal anti-phospho-histone H2AX antibody (Millipore; #05-636). Alexa Fluor 488 from molecular probes (Invitrogen; #A-11034) was used, and fluorescent images were automatically taken for each well of the 96-well plate using an Opera High-Content Screening System (Perkin Elmer). Pictures were taken under non-saturating conditions using a 40x magnification lens to calculate the γ-H2AX nuclear signal intensity.
7
Quantifying DNA Damage Using γ-H2AX
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LCLs were cultured 4 hours before fixation with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, Philadelphia, USA). Two hours before fixation, cells were counted and seeded into a poly-L-lysine-coated (Sigma-Aldrich) μCLEAR bottom 96-well plate (Greiner Bio-One) at a density of 75,000 cells per 100ul full media per well. LCL were then left for 2 hours to attach to the surface of the wells, fixed for 15 min at room temperature, permeabilized in 0.5% Triton X-100 in PBS for 20 minutes at 4°C and stained with primary and secondary antibodies and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) to visualize nuclei. To detect γ-H2AX we used mouse monoclonal anti-phospho-histone H2AX antibody (Millipore; #05-636). Alexa Fluor 488 from molecular probes (Invitrogen; #A-11034) was used, and fluorescent images were automatically taken for each well of the 96-well plate using an Opera High-Content Screening System (Perkin Elmer). Pictures were taken under non-saturating conditions using a 40x magnification lens to calculate the γ-H2AX nuclear signal intensity.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!