Following drug treatments, cells were fixed in 4% formaldehyde for 10 min, washed three times with PBS and permeabilised with 0.1% Triton X‐100 for 5 min. Cells were labelled with Hoechst (final concentration 2 µg·mL−1) for 30 min and finally washed three times with PBS. Hoechst‐labelled nuclei were imaged on a ScanR microscope (Olympus) using a 20× objective, capturing ≥ 4 fields of view per well. Nuclei were classified into different stages of the cell cycle using the ScanR (Olympus, Southend‐on‐Sea, UK) analysis software.
Scanr
Manufactured by Olympus
Sourced in Germany, Japan
The ScanR is a high-performance automated microscopy system designed for screening and analysis applications. The system integrates advanced imaging capabilities, intuitive software, and efficient workflow management to enable comprehensive analysis of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Merck Group (4 mentions)
Hoechst 33258, by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Scanr screening station, by Olympus (2 mentions)
Ix81 fluorescence microscope, by Olympus (2 mentions)
Scanr analysis software, by Olympus (2 mentions)
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
96 well plate, by Greiner (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Lysis buffer, by Qiagen (1 mentions)
Kgm gold, by Lonza (1 mentions)
Hydrogen peroxide, by AppliChem (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Cyclin a, by Santa Cruz Biotechnology (1 mentions)
P21waf1 cip1, by Santa Cruz Biotechnology (1 mentions)
Rad51, by Santa Cruz Biotechnology (1 mentions)
Amd3100, by Merck Group (1 mentions)
41 protocols using scanr
1
Quantifying Cell Cycle Stages
2
Apoptosis Quantification in HTR-8/SVneo Cells
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HTR-8/SVneo cells were cultured at a density of 2x105 cells per well in 12-well culture plates. After 48 h of sPIF treatment, cells were fixed in 4% paraformaldehyde for 20 min at room temperature, washed, and stained using a TUNEL apoptosis kit (In Situ Cell Death Detection Kit, Fluorescein, Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. Cells were counterstained with 1 μg/ml Hoechst33258 (Sigma, St. Louis, MO, USA). Ten fields in each well were imaged using an inverted fluorescence microscope (Olympus, ScanR, Tokyo, Japan), in order to obtain a minimum of 200 cells for analysis. The apoptosis rate was determined by normalizing the number of TUNEL-positive cells against the total number of Hoechst-positive cells, using ScanR software (Olympus, ScanR). Etoposide (42 μM) was used as positive control (data not shown).
3
Quantitative Image Analysis of eYFP-CRAF
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For analysis, the images were subjected to standardized background correction with a 50-pixel window and image segmentation analysis anchored to imaged nuclei as main objects in order to get the total eYFP-CRAF intensity per nucleus and nucleus-associated cytoplasmic region. To eliminate potential image segmentation artifacts, we included in the analysis only the cells where the associated cell segment was smaller than 9,000 pixels—roughly equivalent to the observed cell size on the raw image. We also excluded high-DNA-content artifacts (>4 N), as they predominantly represented unresolved multi-nucleated cell aggregates. Thus, obtained, filtered data were exported and analyzed using Microsoft Excel. All other immunofluorescence experiments imaged and analyzed on the Olympus ScanR were subjected to the same image-analysis protocol.
4
Quantification of PML-NBs and SG DRiP Enrichment
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SGs and PML-NB were quantified in fixed HeLa cells, GFP-PML HeLa Kyoto cells and human iPSCs. The data were imported into the ScanR (Olympus) software. PML-NBs were segmented based on PML signal using edge detection algorithm, except for data shown in Fig. 5B where the SUMO1 signal was used as a proxy for PML-NB segmentation. Nuclei were segmented based on the DAPI signal intensity. PML-NB number/nucleus and PML-NB area was automatically calculated. Concerning DRiP enrichment inside SGs, first, SGs were segmented based on TIA-1 signal using edge detection algorithm. Then, the mean fluorescence intensity of DRiPs was measured in each detected SG and in an area surrounding the SG. The relative enrichment of DRiPs in individual SGs was calculated as a ratio of mean fluorescence intensity inside the SG divided by mean intensity in the region surrounding the SG. The values were plotted as column graphs representing the fraction of SGs with enrichment > 1.5.
Statistical analyses were performed using Student’s t-test for comparisons between two groups or One-way ANOVA, followed by Bonferroni-Holm post-hoc test for comparisons between three or more groups using Daniel’s XL Toolbox or GraphPad Prism6 software.
Statistical analyses were performed using Student’s t-test for comparisons between two groups or One-way ANOVA, followed by Bonferroni-Holm post-hoc test for comparisons between three or more groups using Daniel’s XL Toolbox or GraphPad Prism6 software.
5
Nanoparticle Cytotoxicity Assessment
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Cells were seeded in 48-well plates, and after 24 h, they were treated with the NPs in cell culture media at a concentration of 20 µg/mL for 24 h. ∆Ψm and total ROS were assessed by fluorescence microscopy (IX81 Olympus, Hamburg, Germany) after 30 min incubation in HBSS (GIBCO, Thermo Fisher Scientific, Sant Cugat del Vallés, Spain) with fluorescent probes 5 × 10−6 mol/L tetramethylrhodamine methyl ester (TMRM) and 2’7’–dichlorodihydrofluorescein diacetate (DCFH–DA), and Hoechst 33342 (all probes from Sigma-Aldrich, Madrid, Spain). Cells were washed twice with HBSS and a total of 16–25 images per well were recorded with the fluorescence microscope. The fluorescence signal in individual cells was recorded with a fluorescence microscope coupled with static cytometry software (“ScanR” version 2.03.2, IX81 Olympus). Controls were performed using HBSS, cells in HBSS and NPs solved in HBSS at 1 μg/mL.
6
In Vivo Detection of Pd-Mediated Catalysis
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Wild type zebrafish
embryos were
collected from AB-TPL breeding pairs and reared at 28 °C in E3
embryo medium. Twenty-four hours postfertilization, embryos were treated
with the anesthetic tricaine and pierced in the yolk with a fine needle.
Either nonfunctionalized resin or a Pd0-resin was then
rapidly inserted into the yolk. Embryos that lost significant yolk
in the procedure were removed from the experiment. Embryos were then
gently transferred to fresh E3 medium and returned to 28 °C to
ensure the yolk wound was closed. The corresponding probe (8a,p,b ) was added to the embryo medium (final concentration 5 μM)
and fish incubated for additional 24 h at 31 °C. Fish were imaged
using fluorescent microscopy (Olympus Scan-R). The fold change in
fluorescence with the Pd0 resin +8p in comparison
to that with the inactive resin +8p were quantified
using ImageJ software. A line was drawn horizontally over the yolk
sac of the embryo encompassing both the resin beads and the area surrounding
them (Figure8 b,c), and the pixel intensities
along the lines were calculated (Figure8 d).
Experiments were repeated at least twice with n =
4/per condition. Zebrafish husbandry and experiments were performed
under Home Office License in compliance with the Animals (Scientific
Procedures) Act 1986 and approved by the University of Edinburgh Ethics
Committee.
embryos were
collected from AB-TPL breeding pairs and reared at 28 °C in E3
embryo medium. Twenty-four hours postfertilization, embryos were treated
with the anesthetic tricaine and pierced in the yolk with a fine needle.
Either nonfunctionalized resin or a Pd0-resin was then
rapidly inserted into the yolk. Embryos that lost significant yolk
in the procedure were removed from the experiment. Embryos were then
gently transferred to fresh E3 medium and returned to 28 °C to
ensure the yolk wound was closed. The corresponding probe (
and fish incubated for additional 24 h at 31 °C. Fish were imaged
using fluorescent microscopy (Olympus Scan-R). The fold change in
fluorescence with the Pd0 resin +
to that with the inactive resin +
using ImageJ software. A line was drawn horizontally over the yolk
sac of the embryo encompassing both the resin beads and the area surrounding
them (Figure
along the lines were calculated (Figure
Experiments were repeated at least twice with n =
4/per condition. Zebrafish husbandry and experiments were performed
under Home Office License in compliance with the Animals (Scientific
Procedures) Act 1986 and approved by the University of Edinburgh Ethics
Committee.
7
Enumeration of Circulating Tumor Cells
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Circulating tumor cells (CTC) were successfully enumerated from peripheral blood using maintrac® approach, as reported previously38 (link). In short, 500 ul blood was incubated with lysis buffer (Qiagen, Hilden, Germany) to remove red blood cells, and all nucleated cells were resuspended in 500 ul PBS-EDTA. Subsequently, CTC were stained with 4 µl fluorescently labeled antibodies directed against EpCAM (clone caa7-9G8, Miltenyi Biotec GmbH, Germany) and incubated for 15 min in the cold. Then, the samples were diluted with PBS-EDTA, and a defined volume of the cell suspension and propidium iodide (PI) (Sigma-Aldrich, USA) was transferred to 96 well plates (Greiner Bio-one, USA). Finally, we imaged and enumerated CTC using a fluorescence scanning microscopy (ScanR, Olympus, Tokyo, Japan), enabling visual examination of vital tumor cells. Vital CTCs were defined as EpCAM-positive cells, lacking in nuclear PI staining and with intact morphology, and only these cells were counted. Quality control regarding reagents, instrument standardization, and operator technique was assessed.
8
Evaluating DNA Damage in Keratinocytes
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Primary human keratinocytes were seeded in 96-well plates at a density of 10 × 103 cells/well in KGM Gold (Lonza) and preincubated overnight at 37°C/5% CO2. Cells were then treated with 10 μM and 20 µM DHM for 72 h at 37°C/5% CO2. DNA double strand breaks were induced in positive control wells with 250 µM hydrogen peroxide (AppliChem) for 15 min. Cells were then fixed with formaldehyde followed by immunostaining with Anti-Phospho-H2A.X-AlexaFluor(™)555 antibody (Merck Millipore) and counterstaining with Hoechst 33,342 (ThermoFisher). Image acquisition and analysis was performed on the semi-automated high-content imaging system scanR (Olympus).
9
Transwell Assay for HRMEC Migration
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The migration of HRMECs was assayed using 24-well Transwell chambers with 8-μm pore size filters (Corning, 3422). Briefly, microglia were seeded in the bottom chamber and pretreated for 24 h. Then 1.5 × 104/ml HRMECs were placed into the upper chamber and cocultured at 37 °C under 5% CO2 for 24 h. After fixation with 4% paraformaldehyde for 30 min, HRMECs were stained with 1% crystal violet. The number of the cells migrating to the bottom side of the filter was counted after wiping away the cells on the upper surface. Three images were captured per well under a microscope screening station (scanR, OLYMPUS, Tokyo, Japan). Each experiment was repeated three times.
10
Immunofluorescence Analysis of Cell Signaling
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