Quorum wavefx spinning disk confocal microscope

Cell lines and PDO were plated in LabTek chamber slides and incubated with SiR-DNA stain (Cytoskeleton; 333 nM for cell lines and 1 μM for PDO) for 2 hours before imaging. For live-cell experiments with CFI-402257 treatment, 150 nM CFI-402257 or dimethyl sulfoxide (DMSO) was coadded to the medium with SiR-DNA. Cells were held in a humidified Chamlide stage incubator kept at 37°C and 5% CO₂ (Live Cell Instrument). Time-lapse images were captured using Volocity 6.3 software (Quorum Technologies) on a Quorum WaveFX spinning disk confocal microscope (Quorum Technologies) equipped with a Hamamatsu ImageEM electron-multiplying charge-coupled device camera at ×20 magnification every 4 min for 20 to 24 hours. The time from nuclear envelope breakdown to mitotic exit (i.e., anaphase or chromatin decondensation) was recorded for each dividing cell. For all dividing cells, mitoses were scored as normal, mild segregation errors (i.e., micronuclei, anaphase bridges, and lagging chromosomes), severe segregation errors (i.e., several micronuclei, thick anaphase bridges, asymmetric segregation, multipolar spindles, and multinucleated cells), and mitotic exit without segregation.

Formalin-fixed, paraffin-embedded tissue sections (4 μm thick) were mounted on positively charged microscope slides. Antigen retrieval was performed with microwave heating in EDTA buffer (1 mmol/L EDTA, 0.05% Tween 20 [pH 9.0]). Endogenous peroxidase was blocked using 3% hydrogen peroxide. After blocking for 15 min with blocking buffer in a tyramine signal amplification kit (Alexa Fluor 488 tyramide SuperBoost kit, Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-DDDDK tag (binds to FLAG tag sequence) primary antibody (ab205606, Abcam, Cambridge, UK) was applied overnight at 4°C, followed by the secondary antibody and the fluorescent detection step of the tyramine signal amplification kit. Slides were then washed and mounted with Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA) and immediately imaged on a Quorum WaveFX spinning disk confocal microscope (Quorum Technologies, Puslinch, ON, Canada).