Cells were seeded in 96 well plates and grown to ≈ 50% confluency, transferred to the IncuCyte (Essen BioSciences, Ann Arbor, Mi) after the medium was replaced with fresh medium with or without IT and/or CsA. Membrane damage was measured after adding YoYo-1, a dye that emit fluorescence when it binds to double-stranded DNA. The cytotoxic index is defined as the number of fluorescent objects in a well, divided by the total number of fluorescent objects obtained after 0.1% Triton X-100 is added to open all cells in the well. For migration studies, the wound maker tool was used to make scratch wounds in confluent cell culture monolayers in 96 well image-lock plates (Essen BioSciences). Plates were incubated in the IncuCyte for 24 h and an integrated metric called relative wound density (RWD) was used to quantify effects on migration. This metric measures the cell density in the wound area relative to the cell density outside the wound area.
96 well image lock plate
Manufactured by Sartorius
Sourced in Germany
The 96-well image lock plates are a laboratory equipment product designed for high-throughput imaging applications. These plates feature a specialized well design that helps to minimize image drift and provide consistent sample positioning, enabling accurate and reproducible imaging results.
Automatically generated - may contain errors
Lab products found in correlation
Incucyte scratch wound assay system for 96 well plates, by Sartorius (2 mentions)
96 pin incucyte woundmaker tool, by Sartorius (2 mentions)
Woundmaker, by Sartorius (2 mentions)
Incucyte zoom, by Sartorius (2 mentions)
Incucyte, by Sartorius (1 mentions)
Penicillin and streptomycin, by Merck Group (1 mentions)
Dmem ham s f 12 medium, by Merck Group (1 mentions)
Incucyte zoom microscope, by Sartorius (1 mentions)
Incucyte software, by Sartorius (1 mentions)
Il 10, by Proteintech (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Incucyte imagelock plate, by Sartorius (1 mentions)
Incucyte zoom system, by Sartorius (1 mentions)
Incucyte s3, by Sartorius (1 mentions)
Matrigel coated, by Corning (1 mentions)
Woundmaker tool, by Sartorius (1 mentions)
Rp camps, by Bio-Techne (1 mentions)
Poly l ornithine laminin, by Merck Group (1 mentions)
Incucyte zoom live cell analysis system, by Sartorius (1 mentions)
15 protocols using 96 well image lock plate
1
Evaluating Membrane Damage and Migration
2
Podocyte Monolayer Wound-Healing Assay
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Cells were seeded in 96-well image lock plates (Essen Bioscience, Ann Arbor, MI, USA). Podocytes were examined for confluency as a monolayer via light microscopy before the initiation of the wound-healing assay. Scratches were made using a 96-pin tool (Woundmaker), according to the protocol provided by the manufacturer.
3
Cell Migration Kinetics and Wound Closure
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Cells were plated on 96-well Image-lock plates (Essen Bioscience) and analyzed for confluency as a monolayer via light microscopy before initiation of a scratch wound. Scratches were made using a 96-pin tool (Woundmaker) as per the protocol. Data processing and analysis of migration were performed using the IncuCyte 96-well Kinetic Cell Migration and Invasion Assay software module. Data were then exported to Excel for further analysis. Wound width is defined as the area of the wound at any time t, as determined by the processing software. Wound confluence is expressed as a percentage of the scratch wound that is filled with cells at any given time t, when compared with the initial scratch. Wound closure was monitored at 60-min intervals for at least 48 h. Videomicroscopy was performed with a ×10 objective. Experiments were performed in triplicate at 37 °C.
4
EGFR Mutant LUAD Cell Lines Proliferation
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Two LUAD cell lines, A549 (EGFR wild‐type) and PC9 (EGFR mutant), were purchased from American Type Culture Collection. Cells were cultured in DMEM/Ham's F‐12 medium (Sigma‐Aldrich) for A549 or RPMI‐1640 (Sigma‐Aldrich) for PC9 in a 10 cm culture dish with 10% FBS (Sigma‐Aldrich) and 1% penicillin and streptomycin (Sigma‐Aldrich). The cells were incubated at 37°C in an atmosphere containing 5% CO2. Cells were plated in 96‐well image lock plates (Essen BioScience) at a density of 1 × 103 cells/well for A549 cells or 2 × 103 cells/well for PC9 cells, and incubated at 37°C for 24 h. Later, IL‐10 (Proteintech) or CCL2 (R&D Systems) was added to each well at a concentration of 20 ng/mL. Each well was imaged every 6 h for 72 h under standard incubation conditions using an IncuCyte ZOOM microscope placed in an incubator (Essen BioScience). Image‐based analysis of phase object confluence was performed using the IncuCyte software (Essen BioScience).
5
Quantifying 2D Cell Migration Using IncuCyte
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2D spontaneous migration was monitored using IncuCyte® ZOOM System (Essen BioScience). 1000 cells per well were seeded into 96-well IncuCyte® ImageLock plates (Essen BioScience) and image sets have been collected every 2 h for 72 h. The single cell distance and trajectory have been analyzed using Manual Tracking plug-in for ImageJ (F. Cordelieres, Institute Curie, Paris, France) for 10 cells from three separate experiments. Directionality was calculated as it is described elsewhere (Masuzzo et al., 2017 (link)).
Wound healing assay was performed as described. Cells were seeded onto Essen Bioscience 96-well ImageLock plates and cultured until confluence was reached. With the use of IncuCyte® WoundMaker (96-pin woundmaking tool) unified scratches were made. Collective cell migration was performed with the use of IncuCyte® Live Cell Analysis Imaging System (with data collection every 2 h over 48 h) and analyzed with IncuCyte® software. Results were presented as a percent of scratch overgrown area in time. To establish if Wound Maker is able to remove MatrigelTM from coated surface, a confluent monolayer of cells growing on MatrigelTM coated wells was scratched. Cells and surfaces of wells were immunostained using anti-pan-laminin antibodies and fluorescently labeled phalloidin in order to visualize laminin and F-actin.
Wound healing assay was performed as described. Cells were seeded onto Essen Bioscience 96-well ImageLock plates and cultured until confluence was reached. With the use of IncuCyte® WoundMaker (96-pin woundmaking tool) unified scratches were made. Collective cell migration was performed with the use of IncuCyte® Live Cell Analysis Imaging System (with data collection every 2 h over 48 h) and analyzed with IncuCyte® software. Results were presented as a percent of scratch overgrown area in time. To establish if Wound Maker is able to remove MatrigelTM from coated surface, a confluent monolayer of cells growing on MatrigelTM coated wells was scratched. Cells and surfaces of wells were immunostained using anti-pan-laminin antibodies and fluorescently labeled phalloidin in order to visualize laminin and F-actin.
6
Automated Wound Healing Assay for Cell Migration
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Cells were seeded in 96-well image lock plates (Essen Bioscience) to confluence. After waiting for adherence (4-6h) scratches were made in each well using Incucyte woundmaker tool according to manufacturer instructions. Cells were washed twice with culture media to remove cell debris. Automated time course imaging was performed using Incucyte S3 (Sartorius) in humidified incubator at 37°C with 5% CO2 set to capture each well every hour until wound closure. Analysis was performed using ImageJ software plugin for wound healing analysis.81 (link)
7
Cell Migration Assay with IncuCyte
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Cell migration was determined using the IncuCyte® Scratch Wound Assay system for 96 well plates (Essen Bioscience). 96 well image lock plates (Essen Bioscience) were PLL coated prior to seeding of 40,000 cells per well. Wound application was performed 24 h post seeding using the 96-pin IncuCyte WoundMaker tool (Essen Bioscience) and indicated treatments were applied simultaneously. Measurement was performed in technical replicates (n = 4–8) and repeated as independent, biological experiments as stated for the respective experiment in figure legends.
8
Quantitative Analysis of Cell Migration
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Cells were seeded on 1 mg/mL Matrigel‐coated (Corning) 96‐well ImageLock plates (Essenbioscience). After 24 hours, when the cells reached confluency, standardized wounds were made in all wells simultaneously using Wound Maker™ (Essenbioscience). Phase‐contrast time‐lapse photos were captured using IncuCyte® Live‐Cell Analysis System for 48 hours with a time interval of 2 hours using a 10× objective. An IncuCyte® Scratch Wound Cell Migration Software Module was used for data analysis, and the calculation of relative would density was based on the increase in the area covered by the cells in time. The experiments were performed in triplicate, each condition consisting of four replicates.
For the evaluation of migration distances and cell trajectories, cells were seeded in low density, and images were analysed using ImageJ software with Manual Tracking plugin.20 The distance covered by every cell was measured as the total distance based on the cumulative track lengths. The experiments were performed three times, and each time 40 cells were analysed.
For the evaluation of migration distances and cell trajectories, cells were seeded in low density, and images were analysed using ImageJ software with Manual Tracking plugin.
9
Wound Healing Assay with CCD-25SK Cells
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CCD-25SK cells were seeded in 96-well image-lock plates (Essen BioScience Inc), at a density of 8,000 cells per well in complete growth medium and grown to confluence.
Thereafter, a wound (scratch) was created in each well using a 'Wound Maker' tool (Essen BioScience Inc). This specialized, high precision tool creates a similar size wound in each well of the plate. The wound was then rinsed thoroughly with complete growth medium to remove the detached cells and fresh Si-supplemented medium or controls were added. Phase-contrast images were then acquired every 2 h, over a 96 h period, with the Essen IncuCyte Zoom live cell imaging system. Phase contrast images were analyzed using the IncuCyte Zoom software, first to determine the initial wound area and then to determine the percentage change (increase) in cell confluence within the wounded area at each time point. Each treatment was assessed in at least two independent experiments, with six replicates per treatment per experiment.
Thereafter, a wound (scratch) was created in each well using a 'Wound Maker' tool (Essen BioScience Inc). This specialized, high precision tool creates a similar size wound in each well of the plate. The wound was then rinsed thoroughly with complete growth medium to remove the detached cells and fresh Si-supplemented medium or controls were added. Phase-contrast images were then acquired every 2 h, over a 96 h period, with the Essen IncuCyte Zoom live cell imaging system. Phase contrast images were analyzed using the IncuCyte Zoom software, first to determine the initial wound area and then to determine the percentage change (increase) in cell confluence within the wounded area at each time point. Each treatment was assessed in at least two independent experiments, with six replicates per treatment per experiment.
10
Cell Migration Assay Protocol
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Cell migration was determined using the IncuCyte® Scratch Wound Assay system for 96 well plates (Essen Bioscience). 96 well image lock plates (Essen Bioscience) were PLL coated prior to seeding of 40,000 cells per well. Wound application was performed 24 h post seeding using the 96-pin IncuCyte WoundMaker Tool (Essen Bioscience) and indicated individual treatment conditions were applied simultaneously. Measurement for individual treatment conditions was performed in biological replicates (n = 4 -8) and repeated as independent experiments as stated for the respective experiment (see figure legends).
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