The indicated cell lines were seeded into 96-well plates at a density of 1000–4000 cells per well, depending on the growth rate and design of the experiment. Approximately 24 h later, 10 μM folic acid (T0062, TargetMol), 8 μM p38 MAPK inhibitor SB203580 (T1746, TargetMol), and/or cisplatin were added at the indicated concentrations. Cells were imaged every 3 h using an Incucyte S3 microscope (Essen Bioscience) or Livecyte 419 Phase Focus microscope (Essen Bioscience). Phase-contrast images from the Incucyte S3 microscope were analyzed to evaluate cell proliferation based on cell confluence. Proliferative measurement (cell-doubling time), mitotic measurement (total mitosis events), and kinetic measurements (track speed and displacement) were extracted using Cell Analysis Toolbox (CAT) software available with the Livecyte 419 Phase Focus microscope.
Incucyte s3 microscope
Manufactured by Sartorius
Sourced in Germany
The IncuCyte S3 microscope is a live-cell imaging system designed for continuous monitoring of cell cultures. It provides real-time, quantitative data on cell growth, morphology, and behavior without the need to remove cells from the incubator.
Automatically generated - may contain errors
Lab products found in correlation
Evos fl auto 2 automated microscope, by Thermo Fisher Scientific (2 mentions)
Incucyte 2021a software, by Sartorius (2 mentions)
Dm6 microscope, by Leica (1 mentions)
Cellevent caspase 3 7 detection reagent, by Thermo Fisher Scientific (1 mentions)
Incucyte zoom, by Sartorius (1 mentions)
Il 1β, by Miltenyi Biotec (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
Incucyte technology, by Sartorius (1 mentions)
Incucyte zoom 10 plan fluor objective, by Sartorius (1 mentions)
10 protocols using incucyte s3 microscope
1
Anticancer Potential of Folic Acid and p38 MAPK Inhibitor
2
Automated Imaging of Cell Death Kinetics
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Images was collected using the STACK assay detailed in Forcina et al. (2017) (link). Images were acquired using the IncuCyte S3 microscope (Essen Biosciences; 1408×1040 pixels, at 1.24 μm/pixel). Acquisition settings for the green channel were ex: 460 ± 20, em: 524 ± 20, acquisition time: 300ms; and red channel were ex:585 ± 20, em: 635 ± 70, acquisition time: 400ms. Imaging was performed using a 10x objective. For all experiments, on Day 0 just prior to drug addition, images were taken of a control plate treated with growth media containing 500 nM SYTOX Green as detailed above. For kinetic analysis, images were acquired every 6–8 hours for every well of each plate for 72 hours. For experiments where kinetic analysis was not used images were collected only at the 72 hour end point.
For some experiments that did not require kinetic analysis, images were acquired using an EVOS FL Auto 2 automated microscope (ThermoFisher Scientific). Images were acquired using a 10x objective (EVOS 10x objective, Cat #: AMEP4681). Sytox images were acquired using a GFP filter cube (EVOS LED Cube, GFP, Cat #: AMEP4651, ex: 470/22, em: 525/50, acquisition time: 13.5ms) Mkate2+ images were acquired using a TexasRed filter cube (EVOS LED Cube TxRed, Cat #: AMEP4655, ex: 585/29, em: 628/ 32, acquisition time: 642.0ms).
For some experiments that did not require kinetic analysis, images were acquired using an EVOS FL Auto 2 automated microscope (ThermoFisher Scientific). Images were acquired using a 10x objective (EVOS 10x objective, Cat #: AMEP4681). Sytox images were acquired using a GFP filter cube (EVOS LED Cube, GFP, Cat #: AMEP4651, ex: 470/22, em: 525/50, acquisition time: 13.5ms) Mkate2+ images were acquired using a TexasRed filter cube (EVOS LED Cube TxRed, Cat #: AMEP4655, ex: 585/29, em: 628/ 32, acquisition time: 642.0ms).
3
Automated Imaging of Cell Death Kinetics
4
Senescence Quantification in Organoids
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Organoids were harvested at day 11, fixed and stained for 4 h with X-Gal solution (pH 6) according to the manufacturer's instructions (Merck Millipore, cat#KAA002RF). Organoids were imaged using a Leica DM6 microscope and blue-stained cells were considered as senescent cells.
The quantification of the SA-β-Gal staining of control and irradiated organoids, DMSO/Mdivi-1-treated organoids and DMSO/MFI8-treated organoids has been performed using ImageJ, calculating the ratio between the blue area (positive area) and total area of the organoids.
For the quantification of the SA-β-Gal of DMSO/UA and DMSO/PMItreated samples, organoids were harvested, fixed and incubated for 30 min in Bafilomycin A1, followed by SPiDER-β-Gal reagent as indicated by the manufacturer's instructions (Dojindo EU GmbH, Munich Germany). The samples were next stained with propidium iodide (1 µg/mL) in PBS-Triton X-100 (0.4 %). Samples were imaged using an IncuCyte® S3 microscope (Essen BioScience). The Green fluorescent intensity (300 ms) and red fluorescent intensity (600 ms) of whole-well scans were measured and the green / red area ratio was used for normalization.
The quantification of the SA-β-Gal staining of control and irradiated organoids, DMSO/Mdivi-1-treated organoids and DMSO/MFI8-treated organoids has been performed using ImageJ, calculating the ratio between the blue area (positive area) and total area of the organoids.
For the quantification of the SA-β-Gal of DMSO/UA and DMSO/PMItreated samples, organoids were harvested, fixed and incubated for 30 min in Bafilomycin A1, followed by SPiDER-β-Gal reagent as indicated by the manufacturer's instructions (Dojindo EU GmbH, Munich Germany). The samples were next stained with propidium iodide (1 µg/mL) in PBS-Triton X-100 (0.4 %). Samples were imaged using an IncuCyte® S3 microscope (Essen BioScience). The Green fluorescent intensity (300 ms) and red fluorescent intensity (600 ms) of whole-well scans were measured and the green / red area ratio was used for normalization.
5
Quantifying Caspase-3/7 Activity in Irradiated Salivary Gland Organoids
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Irradiated and non-irradiated (control) salivary gland organoids were collected and immediately treated with CellEvent™ Caspase-3/7 Detection Reagent (cat#C10423, Thermo Fischer Scientific). Green fluorescence intensity was measured in whole wells after 16 h of incubation post-irradiation using an IncuCyte S3 microscope (Essen Bioscience) under 10X magnification and 300 ms exposure. The average Green Fluorescent Intensity per Object was calculated for each sample. For outlier exclusion an upper fluorescence threshold was calculated based on the mean fluorescent intensity across technical replicates (μ + 1.5 σ).
Caspase-3/7 activity was next calculated relative to control samples.
Caspase-3/7 activity was next calculated relative to control samples.
6
Cell Proliferation Assay with IncuCyte
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To evaluate the cell proliferation, IncuCyte® technology (IncuCyte S3 microscope and IncuCyte 2021A software, Sartorius, Göttingen, Germany) was used to visualize cells in real-time and quantified confluency with the IncuCyte® ZOOM living cell imaging system. Cells were seeded at 2 × 104 cells/cm2 at P3 in a 96-well culture plate and incubated during 24 h in HG-DMEM + 10% FBS at 37 °C under 21% O2 and 5% CO2 atmosphere. After 24 h, cells were treated with CHI-HA formulation (NG) (0.1 and 10 µg/mL) or BR formulations (5 and 30 nM of antagonists) with or without IL-1β (10 ng/mL, Miltenyi Biotec, Bergisch Gladbach, Germany) and diluted into HG-DMEM supplemented with 5% FBS. Then, the plate was placed into the IncuCyte® apparatus, and images of cells were recorded every 2 h for a total duration of 144 h.
7
Long-term Cell Proliferation Monitoring
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To track cell proliferation over longer periods, cells were cultured in an Incucyte S3 microscope (Sartorius, Germany) for the indicated time. Initially, cells were seeded at low density into 96-well plates and grown until entering exponential growth (usually 3-4 days). Thereon, cells were treated as indicated, and media and treatment were renewed every 3 days. In the case of experiments with inducible constructs, expression was induced 3 days prior first drug treatment to establish stable expression conditions. Eight-phase contrast images of every well were acquired every 4 h with 20x magnification. Confluency was analyzed and averaged using the Incucyte S3 software. Each experiment was performed in triplicates and mean confluency was plotted using GraphPad prism.
8
Live Cell Imaging of Doxycycline-Induced Cell Death
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HoxB8 cells were seeded in media with or without 1 µg/ml Doxycycline and 1 µg/ml PI and imaged every 2 h in an Incucyte S3 microscope using the ×10 objective (Sartorius, Göttingen, Germany). The analysis function of the system was used to calculate the red object count (ROC), representing PI + cells per mm², normalized to the area covered by the cells at start of imaging. Number of positive cells at the imaging start (t = 0 h) was subtracted from each time point.
For experiments shown in Fig. 1G,H, HoxB8-PF cells were seeded in media containing 1% methylcellulose in a 35-mm Petri dish at a density of 400.000/ml and treated with or without 1 µg/ml Doxycycline and SPY650-DNA stain. Image acquisition was done with Zeiss Axio Observer microscope with ×63 objective every 10 min in an environmental chamber set to 37 °C. A total of 40-52 cells were randomly selected, and mitotic duration and cell fate were assessed manually using Fiji. The clear appearance of metaphase defined the onset of mitosis.
For experiments shown in Fig. 1G,H, HoxB8-PF cells were seeded in media containing 1% methylcellulose in a 35-mm Petri dish at a density of 400.000/ml and treated with or without 1 µg/ml Doxycycline and SPY650-DNA stain. Image acquisition was done with Zeiss Axio Observer microscope with ×63 objective every 10 min in an environmental chamber set to 37 °C. A total of 40-52 cells were randomly selected, and mitotic duration and cell fate were assessed manually using Fiji. The clear appearance of metaphase defined the onset of mitosis.
9
Quantifying eACs Proliferation Dynamics
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A 96-well plate was seeded with eACs (P2) at 2 × 104 cells/cm2 in eAC amplification medium. After 16 h of culture in normoxia, cells were washed with PBS and 200 µL of treatments (corresponding to the conditions tested, i.e., the control medium and the CMs differentially stored (Figure 1 ) or the CMs primed or not (Figure 7 )) were added to each well. The plate was placed at 37°C in normoxia and monitored for 7 days with the live imaging IncuCyte® technology (IncuCyte S3 microscope and IncuCyte 2021A software, Sartorius; Göttingen, Germany) to assess proliferation rate and cell morphology. Using ImageJ software (ImageJ 1.35c, Wayne Rasband, National Institutes of Health, Bethesda, MD, United States), cells were counted on 3 representative areas of each picture taken after CM addition (200 µL/well) and after 48 h of treatment. The proliferation factor was determined by calculating the ratio between cell number at 48 h and cell number at 0 h. Each condition was tested in triplicate and experiments were reproduced 3 (storage assays) to 5 times (priming assays), depending on the experiment.
10
CaMPARI2 Photoisomerization in Microgravity
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The seal’s adhesion was checked after the flight, and no air bubbles or leakages were detectable. For each parabola-set of cells, one well served as a positive control for photo conversion of CaMPARI2 and CaMPARI2-F391W with 100 µM histamine after the flight. The medium of these eight wells per plate was discarded directly after the flight, and DMEM cell culture medium containing 100 µM histamine was added to the wells. After 15 s, these wells were illuminated with the flight hardware for 8 s, and the wells were filled up with medium to 380 µL again. Subsequently, phase contrast and fluorescence images in the green and red channels were acquired. All plates were scanned on-site with an IncuCyte S3 microscope equipped with an IncuCyte Zoom 10 Plan Fluor objective (Sartorius, Göttingen, Germany). For each well, five non-overlapping regions were imaged.
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