2.5. Organoid Viability Analysis
After the organoids were treated for 7 days, the viability was assessed by Live/Dead imaging and quantitatively validated with ATP Luminescent assay (Cell Titer-Glo 3D®, Promega). Luminescence level is proportional to the number of viable cells, thus reflects the level of activity in an organoid. The assay was conducted per the vendor protocol. Briefly, media was removed from the well and replaced with 200 μL of assay solution (100 μL of CellTiter-Glo® 3D Reagent mixed with 100 μL DMEM). The plate was then mixed vigorously on a plate shaker for 5 minutes at 100 rpm followed by incubation at room temperature for an additional 25 minutes. 180 μL of the mixture from each well was transferred to a white flat bottom Polystyrene 96 well plate. Luminescence was measured using a microplate reader (Varioskan LUX, ThermoFisher).
LIVE/DEAD staining was performed on day 7 of organoid culture. Calcein AM (excitation and emission ~495/515 nm, 1:2000 dilution) and Ethidium Homodimer-1 (EthD-1) (excitation/emission ∼528/617 nm, 1:500 dilution) were dissolved in 200 μL of a PBS and DMEM mixture (1:1), and introduced to each organoid with a 30-minute culture. Fluorescent imaging was performed using Nikon A1R Confocal live cell confocal microscope (CMIF, OSUCCC). Z-Stacks were obtained for each organoid and presented as maximum intensity projections.