Thirty thousand of NOY-1 and NOY-1 CisR cells per well were plated in quadruplicates in ImageLock 96-well plates (Essen BioScience, UK) and let to adhere overnight. Confluent monolayers were wounded with wound making tool (Essen BioScience, UK), washed twice and supplemented with culture medium. Images were taken every two hours for next 48 h in the IncuCyte ZOOM™ Kinetic Imaging System (Essen BioScience, UK). Cell migration was evaluated by IncuCyte ZOOM™ 2013A software (Essen BioScience, UK) based on the relative wound density measurements and expressed as means of three independent experiments run in quadruplicates ± SD.
Wound making tool
Manufactured by Sartorius
Sourced in United Kingdom
The Wound making tool is a lab equipment product designed for creating controlled and reproducible wounds on samples. It serves the core function of generating standardized wounds for research and testing purposes.
Automatically generated - may contain errors
Lab products found in correlation
Incucyte zoom kinetic imaging system, by Sartorius (2 mentions)
Imagelock 96 well plate, by Sartorius (2 mentions)
Incucyte zoom 2013a software, by Sartorius (1 mentions)
Matrigel, by Sartorius (1 mentions)
Incucyte s3, by Sartorius (1 mentions)
Incucyte zoom, by Sartorius (1 mentions)
Matrigel, by Corning (1 mentions)
Prism 9, by GraphPad (1 mentions)
Incucyte scratch wound cell migration and invasion system, by Sartorius (1 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
Incucyte zoom or s3, by Sartorius (1 mentions)
Imagelock plate, by Sartorius (1 mentions)
5 protocols using wound making tool
1
Kinetic Wound Closure Assay
2
Quantitative Wound Healing and Invasion Assay
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ImageLock 96-well plates (Essen Biosciences) were used for wound healing assays. For invasion, plates were precoated with 20 μl of 1% Matrigel (Corning) diluted in medium O.N. at 37°C. Cells were resuspended in complete medium at a density of 7 × 105 cells/ml, and 100 μl was added per well. After 4 hours of incubation at 37°C, cells forming a monolayer were wounded using a wound-making tool (Essen Biosciences), washed twice with PBS to remove debris, and overlaid with either 100 μl of medium (for migration assays) or 50 μl of 25% Matrigel in complete medium, followed by the addition of 100 μl of complete media after incubation at 37°C for 1 hour to allow Matrigel polymerization (for invasion assays). Wound closure was imaged every hour for 4 days using IncuCyte ZOOM or IncuCyte S3 (Essen BioScience). Analysis of relative wound density (RWD) was performed using IncuCyte software. Results were normalized to control using Microsoft Excel 16.53 and presented as RWD at each time point (using R v3.6.2) or, unless otherwise specified, at the time point at which the average RWD of control samples reached 50% of confluence (Tmax1/2) using GraphPad Prism 9.
3
Cell Migration and Invasion Assay
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ImageLock plates were coated with 10% GFRM diluted in medium overnight at 37 °C. Cells were re-suspended in 100 μl medium and plated for 4 h at 37 °C. The resultant monolayer was wounded using a wound making tool (Essen Biosciences), washed twice with medium to remove debris, and overlaid with either 50 μl of 25% GFRM for invasion assays or 100 μl medium for migration assays. After an hour at 37 °C 100 μl medium was added to invasion assays and plates imaged every hour for 4 days using the IncuCyte® ZOOM. The number of independent experiments (n) and technical replicates per experiment is stated in the appropriate figure legend. Results are presented as relative wound density (RWD) for each time point and for the time point at which the average RWD of the control samples is 50% (Tmax1/2). Values are mean ± s.d., p values were calculated using a Student’s 2-tailed t-test and are shown on each graph as follows; n.s. = not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p < 0.0001.
4
Wound Healing Assay with PRI-724
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Thirty thousand of NTERA-2 CisR cells (untreated or pretreated with PRI-724 inhibitor for 72 h) per well were plated in pentaplicates in ECM-coated ImageLock 96-well plates (Essen BioScience, Royston, UK) and let to adhere overnight. Confluent monolayers were wounded with wound making tool (Essen BioScience), washed and supplemented with serum-free culture medium with or without PRI-724 inhibitor (1.25 μM). Cell migration was evaluated by IncuCyte® Scratch Wound Cell Migration and Invasion System and documented by the IncuCyte ZOOM™ kinetic imaging system (Essen BioScience). Results were based on the relative wound density measurements and expressed as means of three independent experiments run in pentaplicates ± SD.
5
Wound Healing Assay in 3D Matrigel
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96-well ImageLock plates (Essen Biosciences) were coated with 10% Growth Factor Reduced Matrigel (GFRM) (BD Biosciences) diluted in medium overnight at 37 °C. 70,000 PC3 cells were re-suspended in 100 μl medium per well and plated for 4 hours at 37 °C. The resultant monolayer was wounded using a wound-making tool (Essen Biosciences), washed twice with medium and overlaid with 25% GFRM (50 μl) for an hour. 100 μl medium was then added and plates imaged every hour for up to 4 days using the IncuCyte® ZOOM or S3 (Essen Biosciences). Growth factors or inhibitors were added, at the concentrations described above, to both the GFRM and to the medium. Incucyte ZOOM Live Cell Analysis System Software 2018A or Incucyte S3 Live Cell Analysis System (2021 A) (Essen Biosciences) were used to calculate Relative Wound Density (RWD) for each well. Results are presented as RWD for the timepoint at which the average RWD of the control samples is 50% (Tmax1/2). Number (n) of independent experiments and replicates/condition are detailed in appropriate Figure Legends. Values, mean ± s.d. p values were calculated using Students t-test (two-tailed) or One-Way ANOVA and are shown on each graph as follows n.s. = not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p < 0.0001.
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