Infinite 2000 pro reader

For the purpose of monitoring growth rate, 5×10³ melanoma cells were seeded in white 96-well clear-bottomed plates (Corning) and growth was monitored every 24h using CellTiter Glo kit (Promega) according to the manufacturer’s recommendations. Luminescence intensity was measured using an Infinite 2000 Pro reader (Tecan). Alternatively, 5 × 10⁵ cells transfected with siRNA were seeded per well, in 6-well plates. After 24h, cells were fixed and stained using a ready-to-use solution of crystal violet (Merck). Plates were pictured using a scanner (Epson). Growth was quantified by solubilization of crystal violet in 10% acetic acid and monitoring optical density at 595nm using a Clariostar spectrophotometer (BMG Labtech).

Migration assays were performed as previously described [49 (link)]. Briefly, cells were collected with Accutase (Merck), resuspended in medium containing 1% FBS, and placed in the upper compartments of Transwell chambers (Corning). Medium containing 10% FBS was placed in the lower compartments and the plates were incubated at 37°C for 24 h. The cells were fixed by addition of 4% paraformaldehyde for 10 min and stained with 0.4% crystal violet in 70% ethanol for 15 min. To quantify migrated cells, crystal violet was solubilized by addition of 10% acetic acid, and the optical density (OD) at 595 nm was measured using an Infinite 2000 Pro reader (Tecan).