ORIS™ Cell Migration Assay

Protocol tips

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	Incubate the seeded plate containing the Oris™ Cell Seeding Stoppers in a humidified chamber (37°C, 5% CO2) for 24 hours (cell line dependent) to permit cell attachment.

Protocol tips
Incubate the seeded plate containing the Oris™ Cell Seeding Stoppers in a humidified chamber (37°C, 5% CO2) for 24 hours (cell line dependent) to permit cell attachment.

Publication protocol

Scratch assay []: Cells were seeded into six-well plates at different concentrations, and plates were then incubated overnight at 37 C in a 5% CO humidified atmosphere. Once a confluent monolayer had formed, a straight wound of approximately 1 cm in length was created in the centre of the well using a sterile P200 pipette tip. After scratching, the media was slowly aspirated and discarded, and wells were washed with growth medium (1 mL) to remove debris and smooth the edge of the scratch. After washing with HBSS, fresh medium was added to the cells with or without compound. The plates were then incubated at 37 C in 5% CO for up to 24 hours. During incubation, plates were periodically monitored using phase-contrast microscopy to determine the time required for the migrating cells to fully close the wound. Measurement of the wound was achieved by measuring the gap width at five points in the centre of the wound using an eyepiece graticule with a micrometer scale. After 24 hours, cells were washed twice with Hanks Balanced Salt Solution (HBSS) and fixed with ice cold methanol at -20° C for 30 minutes. After fixation, cells were then hydrated with two washes in PBS and counter-stained with Harris’s Haematoxylin solution. They were then washed briefly under running tap water, before the excess water was drained for 1 minute and the plates left to dry at room temperature. The resulting plates were viewed under a Nikon dissecting microscope (Nikon, C-D55230, Japan) and digital images were acquired for each sample. The extent of cell migration, expressed as percentage migration, was calculated as follows:

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Manufacturer protocol

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