96 well plate woundmaker tool

Migration and invasion of MM cells was assessed as described before [24 (link)]. Briefly, MSTO-wt and MSTO-shCALB2-IPTG (induced) cells were grown to confluence in 96-well ImageLock plates (Essen Bioscience) pre-coated with a thin layer of 0.1 mg/mL Matrigel Basement Membrane Matrix (Corning, Oneonta, NY, USA, Cat. No.354234). A scratch of about 1 mm was created using the 96 well-plate woundmaker tool (Essen Bioscience) as described by the manufacturer. In the case of the invasion assay, 50 µL of Matrigel (0.6 mg/mL) was added to each well after the scratch. After 30 min incubation, 100 µL of medium was added on top and plates were scanned at a 2 h-frequency using the Incucyte™ system (Essen Bioscience). Images were evaluated with the IncuCyte™ software system (version 2011A, Essen Bioscience).

A ‘scratch wound assay’ was performed in both cases. Cells were grown to confluence in 96-well ImageLock plates (Essen Bioscience Inc., Ann Arbor, Michigan, USA) pre-coated with a thin layer of 0.1 mg/ml Matrigel Basement Membrane Matrix (Corning, Cat. No.354234). A scratch of about 1 mm width was created using the 96 well-plate woundmaker tool (Essen Bioscience) as described by the manufacturer. For the migration/proliferation assay, cells repopulating the scratch area grew directly on the surface of the ImageLock plates. In the case of the invasion assay, 50 μl of 1mg/ml of Matrigel Basement Membrane Matrix (10-times higher concentration as the layer on which cells initially grew; for details see Supplemental Methods) was added to each well after the scratch, and cells were incubated for 30 min. Finally 100 μl of medium was added on top of the cells and plates were scanned at a 2-h frequency using the Incucyte™ Live-cell Imaging System (Essen Bioscience). Images were evaluated with the IncuCyte™ software system. The Transwell invasion assay is described in detail in the Supplemental Methods.