After polymerization of a drop of Matrigel (Corning, #356237) - DMEM medium (1:1), cell migration over the edge of the drop does not occur, and the cells inside the drop die within 2-3 days. The methodology was reproduced from several articles. The incubation time of the drop for first polymerization with cells was 1 hour. The assay was performed in a 24-well plate. In less time, the droplet is washed away by the culture medium. In the center of the drop, the cells first showed morphology, but then also died.
Dear Adam, here is our modified protocol according to Richbart SD. et al "Spherical Invasion Assay: A Novel Method to Measure Invasion of Cancer Cells" :
Day 1. PAN02 cells in DMEM (20% FBS) were mixed with Matrigel (1:1) and seeded in the middle of an 24-well plate (50000, 75000 and 150000 per drop, the volume of one drop was 6 microliters). To solidify drops were incubated on standard conditions for one hour. Then 250 microliters of DMEM with 10% FBS was added to each well. The plate was incubated for 24 hours.
Day 2. After 24 h the medium was aspirated from the well and a second layer of Matrigel with DMEM supplemented with 20% FBS (250 microliters, 1:1) was overlaid over the first layer. The plate was incubated at 37°C for 1 h in a cell culture incubator. After 1 h, 250 µL of DMEM with 10% FBS was added to each well. The chamber slide was leaved at 37°C for 24 h in a cell culture incubator.
Day 3. It must be seen, that cells are clearly invading out radially from the primary Matrigel layer in to the secondary Matrigel layer. The first layer contains a high concentration of PAN02 cells mixed with Matrigel. The second layer contains no cells.
On day 3-11 the cell death was observed in the periphery of the drop, but without any invasion to second layer. In the center of the drop, where the thickness of the drop was greater, the appearance of morphology of the cells was observed.
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