Celltiter glo 3d

To determine the temozolomide (TMZ) dosage with which to perform experiments, a dose‐response experiment using GBM12‐only (2.5 × 10⁵ cells mL^‐1) hydrogels was performed that had been cultured for seven days. Hydrogels were cultured with 0–600 × 10^‐6m temozolomide (TMZ, Calbiochem via Millipore Sigma, Burlington, MA), and 0.47% v/v DMSO (Sigma Aldrich) was used as a vehicle control. Hydrogels were cultured for 48 hours before viability was assessed using CellTiter‐Glo 3D (Promega, Madison, WI). TMZ dosage and treatment period were informed by prior studies in the lab as well as chemotherapy studies performed in other 3D in vitro models.^[13, ^55] Hydrogels were equilibrated for 45 min at room temperature, and media was subsequently removed and replaced with 400 µL per hydrogel of 1:1 CellTiter‐Glo Reagent: Knockout DMEM/F12 with StemPro Neural Supplement. Hydrogels were incubated at room temperature for one hour before the CellTiter:media solution was pipetted in triplicate into white‐walled 96‐well plates (Thermo Fisher Scientific) and luminescence was measured using a Biotek Synergy HT microplate reader (Winooski, VT). Viability was additionally assessed on untreated hydrogels at the beginning of the treatment period in order to calculate GR values according to Hafner et al. (Figure S7, Supporting Information).^[21]

An 8 week old, female, C57BL/6 mouse was sacrificed and small intestine crypts were harvested for organoid isolation and cultured as previously described (O'Rourke et al., 2016 (link)). Human colon organoids were cultured as previously described (Sato et al., 2011 (link)). Organoid cultures were passaged and embedded in 25 μl Growth Factor Reduced Matrigel (Corning, 356231) and plated in triplicates in a 48-well plate. Indicated treatments (small intestine: vehicle, 1 μM LGK974, 1 μM LGK974 +50% WNT3A conditioned media, 1 μM LGK974 +30 nM FLAg; colon: WNT3A conditioned media, removal of Wnt from media, 10 nM FLAg) were added to 250 μl of complete media, added to each well on day of passaging and changed every 2–3 days.
At the endpoint (small intestine: 7 days; colon: 12 days), 150 μl CellTiter-Glo3D (Promega) was added to 150 μl media in each well. Organoids were lysed on a rocking platform for 30 min at room temperature. The luminescence reading was measured in duplicates for 20 μl lysate from each well on the Envision multilabel plate reader. The average luminescence reading for each condition was normalized to the control condition to calculate viability.

Cell viability was estimated after addition of PrestoBlue™ reagent (Life Technologies, Carlsbad, CA, USA) for 3 h, following the supplier protocol. For spheroid viability assays, the viability was estimated after addition of CellTiter‐Glo^® 3D for 1 h, following the supplier protocol (Promega, Madison, USA).

Cell viability was determined using CellTiterGlo 3D (Promega) according to the manufacturer’s instructions. More detailed protocols are discussed in SI Appendix.