Factor reduced matrigel matrix

For the tube formation assay, 50 μL of Matrigel (BD Growth Factor Reduced Matrigel Matrix) was added to each well of a 96-well plate and allowed to polymerize at 37°C for 30 min. Next, cells were resuspended in F12K medium without FBS and inoculated in each well at a concentration of 2 × 10⁴ cells/well. After 4 h, the cell structure on the matrix gel was analyzed by an inverted optical microscope (Nikon). All tubular structures of each well were counted. Only closed tubular networks were calculated. These closed tubular networks represent the degree of angiogenesis in vitro.

Briefly, transwell inserts containing membranes with 8 μm pore size (Nunc) were coated with matrigel (Cat N° 356231, growth factor reduced matrigel matrix, phenol red free, BD Biosciences) as per the manufacturer's instructions. Cells (75,000) were spread over the matrigel (1:5) in 200 μl of medium with no serum, and 800 μl of culture medium with 5% of serum was added to the lower chamber. Cell invasion was studied under the effect of platelets, metformin or both. These treatments were added on the lower chamber of the invasion well. For assessing the number of invaded cells, the filters were fixed with PFA for 30 minutes at room temperature and stained with crystal violet. The filters were mounted on cover slides with Kaiser’s, glycerin-gelatin (Merck). Each experiment was performed in duplicate and was repeated at least 3 times. The inserts were examined using a microscope (Nikon Eclipse E200) with attached camera; photographs of 10 fields at 40X, were obtained per insert, and the number of cells in the underside of the filter was counted. Results were expressed as cell number per field, corresponding to the average of the cells photographed for the correspondent treatment.