4.12. 3D Spheroid Model

Spheroids of CRC cells with PAICS knockdown were established by use of the Cultrex® 3D spheroid BME cell invasion assay (Cat# 3500-096-K, Trevigen, Gaithersburg, MD, USA) as described previously [16]. As per the manufacturer’s instructions, 5 × 10³ cells (45 µL) were seeded in 96-well plates in triplicate with 5 µL of Spheroid Formation ECM followed by centrifugation at 200× g for 3 minutes and incubated in a 37 °C, 5% CO₂ incubator to promote spheroid formation. After 72 hours of incubation, 50 µL of invasion matrix was added to the wells, and, to position spheroids within the invasion matrix toward the middle of the wells, centrifugation was accomplished at 200× g for 3 minutes at 4 °C. After 1 hour, warm medium (100 µL) supplemented with FBS was added. The plates were incubated for 4 days, and images were taken with a 4× objective.