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.