Angiogenesis μ plates

All 3D cultures were done in growth factor-free Matrigel Basement-membrane Matrix (Corning Inc., New York, NY, USA) using 96-well Angiogenesis μ-plates (ibidi GmbH, Munich, Germany) as described before.[38 (link)] “Sandwich” assays in short: bottom wells of cooled Angiogenesis plates were filled with 10 μL 4 mg/mL Matrigel, centrifuged for 20 min 200 g and incubated at 37°C temperature for approximately 30–60 min or until the ECM had polymerized. 1,000–1,500 cells were mixed in 2 mg/mL ECM-medium, and 20 μL of cell suspension was added in each well. The plates were centrifuged for 10 min at 100 g, or until the cells had settled on top of the lower ECM. Finally, the outer wells and side reservoirs were filled with water for humidification, and the culture plates placed into the incubator for 3–4 h or overnight. The following day, 60 μL of cell culture medium were added; refreshed every third day by carefully aspirating the medium. This “sandwich” setting allows almost unifocal alignment of cells and 3D structures in a single optical plane, thus reducing the time required for confocal stack imaging.

For miniaturized 3D cultures, PC-3 or PC-3M cells were plated at 150,000 cells/well in 6-well plates and transfected 24 h later with control or FZD8 siRNAs. After 48 h, 3D cultures were set up by embedding silenced cells between two layers of Matrigel^TM and culturing them for 8–9 days on uncoated 96-well Angiogenesis μ-plates (Ibidi GmbH, Martinsried, Germany). This was done by adding 10 μl of Matrigel^TM/culture medium (1:1) per well, allowing to polymerize at 37 °C for 1 h and then adding 650 cells in 20 μl Matrigel^TM/culture medium (1:4) and incubating at 37 °C for 4 h to polymerize. Finally 60 μl of medium was added on top. Humidity was maintained by adding PBS to surrounding wells. Twelve replicate wells were used for each condition and media were changed every second day. Live cell imaging of the 3D assay plates was performed using an IncuCyte^® system (Essen BioScience, Hertfordshire, UK). Images were captured every 2 h for 8–9 days. To monitor silencing efficiency during 3D culture, siRNA-transfected cells were replated in 12-well plates and RNA extracted at days 4 and 8 and analyzed for gene expression using q-RT-PCR.