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 12 to 24 hours

Protocol tips
Incubate the seeded plate containing the Oris™ Cell Seeding Stoppers in a humidified chamber (37°C, 5% CO2) for 12 to 24 hours

Publication protocol

Cell were seeded into an Oris™ 96-well plate with silicon stoppers in place. Oris™ silicon stoppers were cleaned, sterilized, and re-used up to 5 times will no loss of effectiveness. To reuse, sterile stoppers were firmly inserted into 0.4 mL, 96-well optical bottom plates. Cell density was optimized for each cell type such that after overnight attachment, cells had just reached confluence. Depending on cell type this required 3 × 104–6 × 104 cell per well. After attachment, stoppers were removed according to the manufacturer’s protocol, growth medium was aspirated and cells were labeled with CellTrace for 20 minutes at 37 °C. CellTrace Violet (CV) was used at 20 μM and CellTrace Far Red (CFR) was used at 4 μM in Hank’s Balanced Salt Solution (HBSS). After staining, cells were returned to fresh growth medium and imaged immediately then returned to the cell culture incubator for the migration period, which ranged from 12–24 hours, depending on cell type. Migration time was optimized for each experimental cell panel such that cells with the highest migration rate did not completely fill the exclusion zone and cells with the slowest migration rate had sufficient numbers of cells in the exclusion zone that they could be reproducibly detected above background, which is the signal at time point t0. At the end of the migration period, cells were imaged to collect the final image, tf.

Where indicated, cells were treated with 10 μg/mL mitomycin C for 2 hours before stoppers were removed. Where cytochalasin D was used, 1 μg/mL was added to the cell medium at the start of the migration period, immediately after labeling with CellTrace. For serum stimulation experiments, most cells were incubated in serum-free medium for 12 hours prior to initiation of migration, then switched to normal growth medium containing 10% fetal bovine serum (FBS). For EPC2 and CP-A cells, which are adapted to serum-free growth medium, cells were switched to growth factor-free medium for 12 hours before migration. Then migration was stimulated by switching them to normal growth medium additionally supplemented with 2% FBS. For cell migration experiments on collagen, migration wells were coated with a solution of 0.03 mg/mL type I collagen overnight at 37 °C. The solution was then aspirated and wells were rinsed with distilled water and allowed to air dry. Collagen was prepared from a 0.1 M acetic acid stock solution by dilution with PBS.

Conventional, 96-well cell migration assays were also done using the Oris™ migration kit according to the manufacturer’s protocol. After migration, cells were labeled with SYTO 24 for 20 minutes at a concentration of 500 nM in HBSS. The combined effects of migration and proliferation were evaluated by calculating the sum of fluorescent signal detected in the exclusion zone relative to the total signal in the well.

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Manufacturer protocol

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