96 well woundmaker

Cell invasion rates were established using our previously published method
[18 (link),19 (link)]. Using a real-time cell imaging system (IncuCyte™ (Essen BioScience, Michigan, USA). In brief, 96-well plates were coated with a thin layer of collagen by transferring 300 μg/ml of collagen type I (Life Technologies™, Carlsbad, CA) and incubating at 37°C for 30 min. OVCAR-3 and SKOV-3 (2 × 10⁴ cells per well) were grown to confluence in complete growth media. Cell-free zones were created by generating a wound with a 96-Well WoundMaker™ (Essen BioScience, Michigan, USA). The cells were overlaid with 3 mg/ml collagen type I (Life Technologies™, Carlsbad, CA) and incubated at 37°C for 30 min to create a 3D matrix. Complete growth media was added on top of the layer of collagen. Cells were imaged automatically every 3 h over a time period of 48 h. The images were processed by the IncuCyte™ software package (Essen BioScience, Michigan, USA) to measure cell invasion by obtaining the Relative Wound Density (RWD, as defined by custom algorithms within the IncuCyte™ software package). These users informed algorithms identify the wound region and provide visual representations of the segmentation parameters. Image collection was created using three to five representative phase contrast images.

In order to induce a wound in a monolayer of CRC cells, a culture insert (IBIDI, Martinsried, Germany), consisting of two reservoirs separated by a 500-μm thick wall, was placed into each well of six-well plates, and 5 × 10⁴ cells were added into the two reservoirs. After 24 h of incubation at 37°C, the insert was gently removed, and the cells were allowed to migrate for 5 days. Images were taken every 24 h using Olympus IX71 inverted microscope equipped with a digital camera (Olympus France, Rungis, France).
For the comparative analysis of migratory potential according to LOXL2 expression in SW480 cells, control SW480 cells and LOXL2-silenced SW480 cells (5 × 10⁴ cells/well) were seeded into 96-well Essen ImageLock microplates (Essen BioScience, Ann Arbor, MI, USA), and incubated at 37°C for 18 h. Cell monolayers were scratched using a 96-well Wound Maker (Essen BioScience), and the wells were washed with phosphate-buffered saline (PBS) to remove the detached cells. Images of the wounds were automatically recorded at every 6 h for 96 h using IncuCyte ZOOM software (Essen BioScience).

MC38 or SW480 cells (40X10⁴ cells) were seeded in 96 well plates in 100µl DMEM supplemented with10% FCS, 1% PS, and 1% Non-essential amino acids (NEAA) and incubated in 37⁰C, 5% CO2. sCD300b (10µg/ml, Biolegend, San Diego, CA) or hIgG (Bioxcell, lot 659518N1, Lebanon, NH.) were added for 12 hours or until the confluence was greater than 95%. Thereafter a scratch wound was made using 96-well wound-maker (ESSEN Bioscience Inc., Ann Arbor, MI). Subsequently, the cells were washed twice with PBS and 10µg/ml sCD300b or hIgG1 in 200µl Serum and NEAA free DMEM medium ware added. The wound healing were quantified using IncuCyte.

For migration assays, cells were seeded into ImageLock 96‐well Plates (Essen Biosciences) and maintained until 70% confluent. The 700–800 μm scratch wounds were made in each well using the 96 well WoundMaker (Essen Biosciences) directly prior to drug treatment. Plates were imaged every 2 h and migration into the wound area was quantified using the inbuilt Scratch Wound algorithm (Essen Biosciences), adjusted to the individual morphology of each culture type.

Cell mobility was assessed using a scratch wound assay. PC3 cells were cultured in a 96-well plate until it was confluent. The cell layer was wounded using a 96-Well WoundMaker (Essen BioScience, Michigan, USA) and washed twice with fresh serum free media. The cells were incubated with serum free medium, and images of the wounds were automatically taken every 2 h for 48 h using the IncuCyte ZOOM (Essen BioScience). The images were analyzed by the IncuCyte software package (Essen BioScience).