Evos fl auto imaging system microscope

Tumor tissues were enzymatically digested and processed as described previously [81 (link)]. Briefly, freshly harvested tissues were digested with 0.012% (w/v) collagenase XI (Sigma) and 0.012% (w/v) dispase (GIBCO, MD, USA) in DMEM media containing 1% FBS (GIBCO) and embedded in growth factor reduced Matrigel (BD Biosciences, San Jose, CA, USA). The organoids were cultured in AdDMEM/F12 (GIBCO) media supplemented with 0.1% insulin–transferrin–selenium (ITS-G) (100×) (Gibco™, MD, USA), FGF10, and FGF 2 (PreproTech, Cranbury, NJ, USA), in a 5% CO₂ incubator at 37 °C. On the 5th day, the tumor organoids were treated with drugs, and changes in the size and morphology were followed consecutively for 7 days. The bright-field images were acquired on an EVOS^® FL auto imaging system microscope (Life Technologies, CA, USA), followed by quantification of the change in area of selected organoids.

The trans-endothelial migration (TEM) was performed as described earlier [76 (link)]. Briefly, we seeded 5 × 10⁵ brain endothelial cells on the upper outer surface of the gelatin coated trans-well for 24 h. In the lower chamber, 5 × 10⁵ human astrocytes were cultured for 24 h. the next day, drug treated brain tropic SKBrM3 cells were seeded onto the top of endothelial cells in a trans-well insert. All the three cells types were incubated for the next 16 h in co-culture conditions, and transmigrated cells were counted by fixing cells in 100% methanol and stained with 0.1% crystal violet. Transmigrated cells were imaged and counted using an EVOS^® FL auto imaging system microscope (Life Technologies, CA, USA).