300 inverted fluorescence microscope

Uveal melanoma spheroids were prepared and placed in a collagen matrix as previously described (Smalley, Contractor, et al., 2008a (link); Smalley et al., 2006 (link)). The spheroids were treated with drugs for 72 hr, and subsequently stained with Live/Dead viability stain (Thermo Fisher, Carlsbad, CA) to be analyzed using a Nikon-300 inverted fluorescence microscope (Nikon, Melville, NY). Red puncta, indicating dead cells, were measured for each spheroid.

Melanoma three-dimensional (3D) spheroids were prepared as described previously (34 (link)). Briefly, 50 μL of a 1.5% solution of agarose was added to each well of a 96-well tissue culture plate and allowed to solidify. Into each well, 5×10³ cells in 200 μL of media (containing the respective compound that the cells are resistant to) were overlayed on the agarose bed and allowed to grow over 5 days. The resultant spheroids were implanted into rat tail collagen I and treated for 6 days with either 1 μM vemurafenib alone or in combination with AT13387 (single agent resistant cell lines) or 1 μM vemurafenib and 1 μM selumetinib alone or in combination with AT13387 (dual agent resistant cell lines). Spheroids were then washed three times in media and stained with calcein-AM and ethidium bromide (Molecular Probes) for 1 hour at 37 °C, according to the manufacturer's instructions. Pictures of the invading spheroids were taken with a 5x objective using a Nikon 300 inverted fluorescence microscope. Average spheroid area was quantified using ImageJ 1.46r software.