Multicellular tumor spheroids (MCTS) were formed using the liquid overlay method [19 (link)], collected and fixed in 4% paraformaldehyde/PBS. For cryopreservation fixed MCTS were dehydrated with sucrose and snap-frozen with Optimal Cutting Temperature media (Tissue Tek, Torrance, CA). 5 μm cryo-sections were blocked in PBS pH 7.4, 0.15% triton-X 100 vv−1, 10% FBS vv−1, and 2% BSA wv−1 in a humidified chamber, incubated with the indicated antibodies. DNA was labeled with Hoechst dye. Sections were mounted using Fluorogel with DABCO (17985-04; Electron Microscopy Science, Hatfield, PA). Fluorescently stained MCTS sections were imaged on a Zeiss confocal microscope. Nuclei were counted as Hoechst-positive cells by using watershed separation and quantification with particle analysis in ImageJ software (ImageJ, RRID:SCR_003070) followed by manual counting of the stained nuclei.
4 × 104 PDX cells were seed in 96 wells plate pre-coated with 1% agarose in 100 μL DMEM with 10% FBS. Spheroids were monitored and measured. After treated with BZ for 96 hours, spheroids were fixed in 4% PFA for 30 min and cryoprotected in 15%, 30% sucrose and embedded in O.C.T. and 5 μm cryostat sections were cut.
4 × 104 PDX cells were seed in 96 wells plate pre-coated with 1% agarose in 100 μL DMEM with 10% FBS. Spheroids were monitored and measured. After treated with BZ for 96 hours, spheroids were fixed in 4% PFA for 30 min and cryoprotected in 15%, 30% sucrose and embedded in O.C.T. and 5 μm cryostat sections were cut.
