Oris™ Cell Migration Assay - Fibronectin Coated

Protocol tips

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	- Cells are washed after 24h incubation with pre-warmed, serum- and phenol-red-free DMEM (EGF and OPN were dissolved in DMEM+1% FBS v/v+1% v/v penicillin / streptomycin). After another 24h, wells are washed in DMEM without phenol red and stained with 2 µM SYTO 24® DNA stain and 3 µM propidium iodide for 30 min at 37 °C

Protocol tips
- Cells are washed after 24h incubation with pre-warmed, serum- and phenol-red-free DMEM (EGF and OPN were dissolved in DMEM+1% FBS v/v+1% v/v penicillin / streptomycin). After another 24h, wells are washed in DMEM without phenol red and stained with 2 µM SYTO 24® DNA stain and 3 µM propidium iodide for 30 min at 37 °C

Publication protocol

"FHs-74 int cells were cultured using DMEM+10% v/v FBS+1% v/v penicillin/streptomycin+10 ng/ml recombinant human insulin at 37 °C 5% CO2 and sub-cultured three times a week at 1:20. The FHs-74 int cells were monitored for morphological changes and experiments carried out between passage three and 15. Caco-2 cells were cultured using DMEM+10% v/v FBS+1% v/v penicillin/streptomycin at 37 °C 5% CO2 and sub-cultured three times a week at 1:10. Experiments were carried out between passage three and 25.

The migration rate was quantified using a modified assay by Platypus technologies. The silicone inserts were rinsed in 70% ethanol, evaporated dry, washed in growth media and mounted in a clear bottom, 96-well plate coated with 50 µg/ml BME protein as per the “thin gel” manufacturer specification. The outermost rows and columns were avoided due to rim effects. 100,000 cells were seeded per well around the inserts to reach rapid confluence. After a 24 h incubation period, the inserts were removed and cell debris gently removed by washing once with pre-warmed, serum- and phenol-red-free DMEM. EGF and OPN were dissolved in DMEM+1% FBS v/v+1% v/v penicillin/streptomycin and gently added by dispensing down the well wall. The FBS concentration in the starvation media was found by titrating FBS concentration versus cellular morphology changes and proliferation/apoptosis in the assay (Supplemental Fig. 1A and B). Upon another 24 h incubation the plate was washed in DMEM without phenol red and stained with 2 µM SYTO-24® DNA stain and 3 µM propidium iodide for 30 min at 37 °C. Experiments focusing on morphology were done without propidium iodide, but with 5 µg/ml CellMask orange added at the last 5 min of the incubation as recommended by the manufacturer. Following incubation the wells were washed once in pre-warmed, serum- and phenol-red free DMEM before adding 100 µl pre-warmed, serum and phenol-red free DMEM+25 mM HEPES to stabilize the monolayer while acquiring images"


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