Essen imagelock plate

Cell migration assays were conducted using an IncuCyte 96‐Well Real‐Time Cell Migration System (Essen Bioscience, USA). Cells (1 × 10⁵/well) were seeded into 96‐well Essen ImageLock plates (Essen Bioscience, USA) and grown to almost 100% confluence. A 96‐pin Wound Maker was used to create homogeneous scratch wounds through a confluent cell monolayer. The plate was washed with cold phosphate‐buffered saline (PBS) and scanned every hour for 24 h. The data were assessed based on the relative wound density, and images were obtained via phase‐contrast imaging. Data were analysed using GraphPad Prism software.

For migration, cells were seeded in triplicate on 96-well Essen Image Lock plates (4379, Essen BioScience, Ann Arbor, MI, USA) at 80 × 10³ per well with 100 μL medium and were allowed to adhere overnight. After wounding with the wound maker (Essen BioScience, Ann Arbor, USA), the medium was aspirated, and each well was washed two times with PBS followed by addition of 100 μL of medium. Analysis of wound closure was performed using the Incucyte Scratch Wound Analysis module. Images were obtained every 2 h. For proliferation, cells were also plated in triplicate on 96-well plate (3595, Essen BioScience) at 1 × 10³ per well with 100 μL of medium and were allowed to adhere overnight. The Incucyte Standard module was applied for proliferation analysis, and images were acquired every 2 h.

Prior to initiating the migration assay, 1×10⁴ cells per well were seeded in 96-well Essen ImageLock plates (Essen Bioscience, Ann Arbor, Michigan, USA) and grown for 48 h to confluence under standard conditions. Then, cell-free zones were generated by using a 96-pin WoundMaker (Essen Bioscience, Ann Arbor, Ml) to simultaneously create wounds in all wells.
Thereafter, the plates were placed inside an automated microscope (IncuCyte™ (Essen Instruments)) which resides inside a standard cell culture incubator and equilibrated for 2 h before the first scan.
The cells were scanned every 3 h and the width of the cell-free zone was determined by IncuCyte™ software which is capable to identify the exact wound region.
To calculate the exact distance of cell migration the detected wound width of each time point was subtracted from the first measured wound width in the corresponding well. This value was divided by 2 and indicates the length (μm) of migration.
In parallel pictures were taken by IncuCyte from each well at every time point. The experiment was performed in triplicate.

The cells were seeded in 96-well Essen ImageLock plates (Essen BioScience) to achieve a confluent density (∼5 × 10⁵ per well). After 24 h, cells were treated with mitomycin C (Roche Catalog # 10107409001) for 3 h and scratch wounds were made simultaneously in all culture wells using an Essen WoundMaker. For the Invasion assay using Incucyte, wells were coated with 100 µg/mL basement membrane extract (Cultrex, Trevigen-3433-010-01) in DMEM overnight before cell seeding and, after wound creation, wells were washed to remove dislodged cells and 50 µL of 1 mg/mL of reduced growth factor basement membrane extract diluted in culture mediamedium was added to fill the wound with extra cellular matrix (ECM). The plate was placed in a 37 °C humidified incubator for 1 h to allow the basement membrane to settle, then 50 µL of culture media ±20 ng/mL EGF was added so that the final concentration added was 10 ng/mL. The plates were scanned in the IncuCyte live-cell imaging system (Essen BioScience) at 2-h intervals for 72 h. The data were analyzed with the IncuCyte scratch wound assay software module (Cat No. 9600-0012) and version 2014A.

For in vitro migration studies, cells were plated in triplicate on 96-well Essen Image Lock plates (Essen BioScience) at 40,000 cells per well. Cancer cells were pre-treated with 30 ng/ml Mitomycin C for 2 h to block cell proliferation. The Essen Wound Maker (Essen BioScience) was used to make the wounds in confluent cell culture monolayers. Cancer cells were then treated with either 25 or 50 μg ml⁻¹of collagen I (Invitrogen) after scratch. Wound closure was monitored by acquiring images every 1 h over a 24 h period with the Incucyte ZOOM. An integrated metric called relative wound density (RWD) was used to quantify effects on migration. The grey area indicates the “wound gap” (devoid of cells), while the orange area indicates the movement of cancer cells migrating toward the gap area.

The modulation of cell migration was analyzed by wound-healing assays. Briefly, NHDFs were seeded in a 96-well Essen ImageLock plate (Essen BioScience) and were grown to confluence. After 24 h, the scratches were made using the 96-pin WoundMaker (Essen BioScience), followed by incubation of the cultures in media with 10 ng/ml of mitomycin C to block cell proliferation. VCE-004.8 and TGFβ1 or rhIL-4 (10 ng/ml) were added and wound images were taken every 60 min for 36 h, and the data analyzed by the integrated metric Relative Wound Density part of the live content cell imaging system IncuCyte HD (Essen BioScience).