High content screening of strain collections was performed using an automated microscopy set-up (ScanR system, Olympus) as previously described 26 (link). Images were acquired using a 60× air lens using GFP (Excitation 490/20 nm, Emission 535/50 nm), mCherry (Excitation 572/35 nm, Emission 632/60 nm), and bright-field channels. When a cytosolic mCherry cell marker was used (Fig. 2 ), images were analyzed using the ScanR Analysis software (Olympus), and single cells were recognized based on the mCherry channel. Measures of cell size, shape and fluorescent signals were extracted. For localization assignments, images were manually reviewed using ImageJ. Since no co-localization markers were used we only assigned localizations that could be easily discriminated by eye: ER, nuclear periphery, cytosol, cell periphery, vacuole lumen, vacuole membrane, mitochondria, nucleus, bud/bud neck and punctate (that would include the Golgi apparatus, peroxisomes, endosomes, other vesicular structures and subdomain compartments) (Fig. 3c ).
Scanr system
Manufactured by Olympus
Sourced in Germany
The ScanR system is a high-speed, automated digital imaging system designed for microscopy applications. It captures and analyzes images of biological samples with precision and efficiency.
Automatically generated - may contain errors
Lab products found in correlation
Scanr analysis software, by Olympus (5 mentions)
Orca r2, by Hamamatsu Photonics (3 mentions)
Scanr analysis software 2.7.0 r3429 x64, by Olympus (2 mentions)
Dmi6000 microscope, by Leica camera (2 mentions)
Orca flash4.0 lt camera, by Hamamatsu Photonics (2 mentions)
Plan apochromat 63 1 and 100 1, by Hamamatsu Photonics (2 mentions)
Axio observer, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Prism 8, by GraphPad (1 mentions)
Oil objective, by Leica camera (1 mentions)
Antibody against γh2ax, by Cell Signaling Technology (1 mentions)
Las af lite software, by Leica camera (1 mentions)
Leica sp8 confocal microscope, by Leica camera (1 mentions)
Rabbit anti human p65a, by Santa Cruz Biotechnology (1 mentions)
Normal rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
D2650, by Merck Group (1 mentions)
C10600, by Hamamatsu Photonics (1 mentions)
Rotor bench top colony arrayer, by Singer Instruments (1 mentions)
29 protocols using scanr system
1
High-Content Screening of Yeast Strains
2
Automated Multichannel Microscopy for QIBC
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Automated multichannel wide-field microscopy for high-content imaging and quantitative image-based cytometry (QIBC) was performed using the Olympus ScanR System as described previously (Kilic et al., 2019 (link); Teloni et al., 2019 (link)). Images were analyzed with the inbuilt Olympus ScanR Image Analysis Software Version 3.0.1, a dynamic background correction was applied, and nuclei segmentation was performed using an integrated intensity-based object detection module based on the DAPI signal. All downstream analyses were focused on properly detected nuclei containing a 2C-4C DNA content as measured by total and mean DAPI intensities. Fluorescence intensities were quantified and are depicted as arbitrary units. Color-coded scatterplots of asynchronous cell populations were generated with Spotfire data visualization software (TIBCO) and GraphPad Prism 8.0 was used for depicting data as violin plots. Within one experiment, similar cell numbers were compared for the different conditions. For visualizing discrete data in scatterplots, mild jittering (random displacement of data points along discrete data axes) was applied in order to demerge overlapping data points. Representative scatterplots and quantifications of independent experiments, typically containing several thousand cells each, are shown.
3
Automated Yeast Cell Imaging Workflow
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Samples along the depletion time course were fixed in 1% formaldehyde for 15 min and quenched in 125 mM Gly. Bright-field images were automatically collected in multiple fields per sample along an 8 μm z-stack (1μm step size) using a Scan^R system (Olympus). Cells were segmented using freely available yeast segmentation software (66 (link)).
4
High-Content Screening of Yeast Strains
5
Immunofluorescence Analysis of DNA Damage Response
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Cells grown on coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.5% Triton X1−00 for 10 min. Cells were further incubated with ice-cold methanol for 5 min and blocked with 3% BSA in PBS for 30 min. Coverslips were incubated with primary antibodies for 3 h, washed with PBS, and incubated with AlexaFluor-conjugated secondary antibodies for 1 h. Mounting was performed using Vectashield. Imaging was performed using Leica Sp8 confocal microscope equipped with 63× oil objective (NA 1.40). Images were analyzed using LAS AF Lite software (Leica, Wetzlar, Germany). Induction of DNA damage response was evaluated as described previously [32 (link)]. Briefly, cells were exposed to ionizing radiation (3 Gy) using X-RAD 225XL instrument (Precision; Cu filter 0.5 mm), fixed with 4% PFA, permeabilized with 0.5% Triton X1−00, and probed with antibody against γH2AX (Cell Signaling Technology). Images were acquired using Olympus ScanR system equipped with 40×/NA 1.3 objective (Olympus, Tokio, JApan). Number of γH2AX-positive foci per nucleus was determined using spot detection module. More than 300 nuclei were quantified per condition.
6
Quantifying Transcription Factor Translocation
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Immunofluorescence staining of TLR8 and transcription factors and quantification of nuclear accumulation by high content screening (Scan^R system, Olympus) was done as previously described (24 (link)). The following antibodies were used: mouse antihuman IRF5 mAb (Abcam, #10T1), rabbit antihuman IRF3 XP mAb [Cell Signaling Technology (CST), no. 11904], rabbit antihuman p65/RelA XP mAb (CST, no. 8242), rabbit antihuman p65 A (Santa Cruz Biotechnology, no. sc-109), rabbit antihuman IRF1 XP mAb (CST, no. 8478), and rabbit antihuman TLR8 XP mAb (CST, no. D3Z6J). For TLR4 IF staining monocytes were washed by ice-cold PBS twice, fixed by cold methanol-acetone (1:1) at −20°C overnight, rehydrated in PBS for 1 h, and blocked by 20% human serum in PBS for 30 min. After blocking, cells were incubated with primary antibodies diluted in 2% human serum [2 µg/ml rabbit anti-TLR4 IgG (H-80) or normal rabbit IgG (Santa Cruz Biotechnology)] at +4°C overnight, washed three times by PBS with 2% human serum, incubated with secondary A488-labeled goat antirabbit IgG (Life Technologies). After three washes, cells were left in PBS at +4°C prior to confocal microscopy imaging, which was done using Leica SP8 with a 63× objective (NA 1.4).
7
Multiparametric Fluorescent Assay for Cell Toxicity
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Following treatments, cells were simultaneously loaded with several fluorescent dyes to measure multiple biomarkers of cell toxicity. Different combinations of fluorescent probes were used to identify specific mechanisms of toxicity according to previously described HCS assays (Donato et al. 2012 (link); Tolosa et al. 2015 (link), 2012b (link)). Information about the probes is summarised in Supplementary Table S2.
After incubating with dyes, cells were imaged by the Scan^R system (Olympus, Germany). Dyes were excited, and their fluorescence was monitored at the excitation and emission wavelengths at appropriate filter settings. The collected images were analysed using the Scan^R analysis module, which allows the simultaneous quantification of subcellular structures that are stained by different fluorescent probes. The measured fluorescence intensity was associated with the predefined nuclear and cytoplasmic compartments (Tolosa et al. 2012b (link)).
After incubating with dyes, cells were imaged by the Scan^R system (Olympus, Germany). Dyes were excited, and their fluorescence was monitored at the excitation and emission wavelengths at appropriate filter settings. The collected images were analysed using the Scan^R analysis module, which allows the simultaneous quantification of subcellular structures that are stained by different fluorescent probes. The measured fluorescence intensity was associated with the predefined nuclear and cytoplasmic compartments (Tolosa et al. 2012b (link)).
8
Automated Microscopy Screening Workflow
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Microscopic screening was performed using an automated microscopy system (ScanR system; Olympus) as previously described (24 (link)). Briefly, images were acquired for GFP (excitation 490/20 nm; emission 535/50 nm) and bright-field channels. After acquisition, the images were manually reviewed in MATLAB vs.2012a 7.17 using compare2picturesV5 script. As there were no co-localization markers, we assigned only those localizations that could be easily discriminated by eyes.
9
Automated Single-Cell Fluorescence Imaging
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Single cell fluorescence measurements were performed using an automated microscope system as described in Cohen and Schuldiner, Methods Mol. Biol. 781, 127–59 (2011). Briefly, strains were cultured over-night and diluted in the same manner as in the microplate reader measurements. Following an incubation of four hours in 30°c in a shaking incubator (LiCONiC Instruments), cells were then transferred onto glass bottom 384-well microscope plates (Matrical Bioscience) coated with Concanavalin A (Sigma-Aldrich). The microscope plates were conveyed to an automated inverted fluorescent microscopic ScanR system (Olympus), equipped with a cooled CCD camera. Images were acquired using a 60× air lens using YFP (E.x. 490/20 nm, E.m 535/50 nm), mCherry (E.x. 572/35 nm, E.m 632/60 nm), and bright-field channels. After acquisition images were analyzed using the ScanR Analysis software (Olympus), and single cells were recognized based on the mCherry channel. Measures of cell size, shape and fluorescent signals were extracted. The top and bottom scoring single cells in terms of cell size and shape within each strain were gated out of further analysis to ensure homogenous and correct cell recognition, yielding a mean of 435±164 cells analyzed per strain (minimum of 69 cells).
10
Immunofluorescence microscopy for DNA damage response
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For indirect immunofluorescence microscopy, cells were cultured and treated where indicated with 10 μM CPT (C9911, Sigma) for 45 min, irradiated (X-RAD 225XL, Accella) with 20 Gy of X-rays on ice or 2 Gy of X-rays at RT in the presence of DMSO (D2650, Sigma), 10 μM PARG inhibitor (PDD 0017273; 5952, Tocris Bioscience) or 5 μM DNA-PK inhibitor (NU 7441; 3712, Tocris Bioscience). Cells were fixed with 4% formaldehyde and immunostained as described previously (7 (link)). Images were taken using a DMi6000 microscope (Leica) with 40× dry objective. Automated wide-field microscopy was performed on scanR system (Olympus) with scanR Image Acquisition and Analysis Software, 40 x/0.95NA (UPLSAPO 2 40×) dry objective.
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