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Assay procedure - 'Methods'
The fences should be cleaned and sterilised before each assay. While not in use the fences should be kept up-side-down on the metal side to protect silicone washers. Use a detergent, ethanol or a short-time ultrasonic bath (1-2 min) to clean the fences. The optimum temperature for sterilisation is 121°C (2 atm, wet). Fences can also be sterilised at 180°C (1 atm, dry). However, this may cause damage to the silicone component in long-term use. Note: if used directly after sterilisation, the very adhesive silicone may lead to damage of surface coatings and the silicone washer itself. However, using freshly sterilised fences may help to minimise problems with leaky washers.
Before the experiment is started the sterilised supports are inserted into the wells and the plate is placed in an incubator for 30 min to adapt at 37°C and to allow the silicone washers to attach to the surface.
In the 'layer expansion' regime the internal chamber of each 16mm-device is charged with the cells suspended in 150 µl medium, e.g. with 30,000 HUVEC/well to obtain a confluent monolayer. In addition the external chamber of each support is to be charged with 650 µl of medium only, especially if there are problems with leaky washers. In the 'wound closure' regime the outer chamber is loaded with cells (e.g. 130,000 HUVEC in 650 µl) and the inner with 150 µl medium only. Similar cell interaction models may be developed placing one type of cells inside, another type outside the washer. It is important to introduce the tip of the pipette deeply into the internal chamber (2mm above the bottom) in order to avoid air bubbles.
After allowing the cells to attach (about 4-5 hours, depending on the cell type) the supports are removed using forceps and the cell layer is washed twice in order to remove non-adherent cells. Subsequently 1 ml of medium is applied with/without the drugs to be investigated. (Cell must not be cultured over days within the fences, as the liberation of metal ions may influence cell behaviour.)
In order to distinguish between proliferation and migration, at this time the cells can by exposed to irradiation with 15-25 Gy (1500-2500 rad) or to toxins like 5-fluorouracil (10-20 µg/ml; according to literature up to 100 µg/ml) to prevent proliferation.
The diameter at the beginning is about 6.7 mm, corresponding to an area of 35 mm2, depending on the type of cells. The cells are incubated for days (e.g. HUVECs for 5 to 7 days), while the incubation medium is changed every 2 days. After allowing the cell monolayer to expand for days the experiment is terminated by fixing the cells in ethanol:methanol (1:1) or in 5 to 10% paraformaldehyde in buffer for 3 to 10 minutes. After staining (e.g. with 10% Giemsa solution for 20 min followed by alcohol rinsing for 1 min) the cell-covered area is measured under a stereo-microscope (e.g. with help of a mm-graded foil) or is evaluated by a computer-driven video system. Assays should be done in triplicate or quadruplicate.
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