Evaluating Anchorage-Independent Cell Growth

Anchorage-independent growth was assessed as previously described [21 (link),22 (link)]. Briefly, 6-well plates were coated with a base layer of 0.9% low-melting agarose (Biozym Scientific GmbH, Hess. Oldendorf, Germany) containing 10% FBS, antibiotics and the respective reagents or corresponding solvents. A layer of 0.35% agarose containing the same supplements as the base layer plus 2 × 10⁴ cells/well was placed on top of the base layer prior to incubation for 21 d at standard culture conditions. Microscopic images were taken at 4-fold magnification. The largest diameter of the colonies was measured, and colonies with a diameter exceeding 150 µm (T98G, SC35, SC38 and SC40) or 200 µm (U87MG, PC35, PC38 and PC40) were counted.