Fxcycle pi

Cellular DNA content was measured using FxCycle PI (propidium iodide)/RNAse staining solution (Invitrogen, F10797) according to the manufacturer’s instructions. Cyclin B1 quantification was performed in cells fixed with BD fixation/permeabilization solution (BD, 554714, Becton, Dickinson and Company, Heidelberg, Germany) and stained with Cyclin B1-AF647 (Cell Signaling Technology, 4118). The samples were analysed using a BD Accuri C6 Plus flow cytometer (Becton–Dickinson) applying 535/5 nm excitation and emission collected in a 617/20 band-pass. Cells were gated according to their size and granularity; only morphologically intact host cells were included in the analysis. All analyses were performed in FlowJo v.10 software.

To ensure sufficient cell numbers, five tumouroids were pooled and digested
(collagenase type I, 37°C, 1 h) on a shaker (200 IU/mL in Hanks’ Balanced Salt
Solution (HBSS)/calcium/magnesium; Gibco). The digest was centrifuged at
700g for 5 min. The supernatant was discarded, and cells
were incubated in TrypLE Express (Gibco) to obtain a single cell suspension,
washed by centrifugation and resuspended in 250 µL PBS. Ice cold 70% ethanol was
added dropwise (2 mL) to the cell pellet while vortexing and cells stored in the
fridge, overnight. For processing, cells were centrifuged twice at
800g for 5 min, stained using FxCycle PI (Invitrogen) for
20–30 min at room temperature, and flow cytometry analysis performed on a BD
LSRII using FACS DIVA and FlowJo software.

PC3 and 22RV1 cells were seeded in a 10 cm dishes (0.5 × 10⁶–2 × 10⁶/dish), incubated overnight and treated without or with canagliflozin (CANA) (0–30 µM) and or radiotherapy (RT) (0–8 Gy). After treatments, cells were incubated for 48 h until they were 50–60% confluent. Cells were then harvested and washed with cold PBS buffer, fixed in 70% ethanol, and stored at −20 °C. Before analysis cells were centrifuged, EtOH was aspirated, cells were washed with PBS and stained with propidium iodide (ThermoFisher FxCycle PI) used to stain cells for 30 minutes followed by flow cytometry analysis, using a Cytoflex LX flow cytometer (Beckman Coulter, Mississauga, ON) (Core Flow Facility, McMaster University). For the gating strategy, we utilized forward scatter and side scatter to identify viable, single-cell events. This cell population gate was positioned on PI-area vs PI-height to eliminate doublets from the analysis. Data analysis was performed using FlowJo software (Version 10.8.0, FlowJo LLC, Ashland, OR).