Celigo s cell imaging cytometer

Organoids were seeded manually according to our previously published protocols^{3 (link),16 (link),33 (link)}. Briefly, single cells suspended in a 3:4 mixture of Mammocult (StemCell Technologies 05620) and Matrigel (Corning 354234) were deposited around the perimeter of the wells of either 24-well or 96-well plates. The cell suspension was kept on ice throughout the seeding process to prevent the gelation of the Matrigel. To seed organoids in a 96-well plate (Corning 3603), a pipette was used to distribute 5 µL of cell suspension (5 × 10⁵ cells/mL) along the bottom perimeter of each well. Once all mini-rings were generated, plates were incubated at 37 °C and 5% CO₂ for 20 minutes to solidify the Matrigel, and 100 µL of pre-warmed Mammocult medium was added to the center of each well using an epMotion 96 liquid handler (Eppendorf). To generate larger rings (maxi-rings) in 24-well plates (Corning 3527), 70 µL of cell suspension (1.4 × 10⁶ cells/mL) was deposited around the perimeter of each well. Following seeding, the plate was incubated at 37 °C and 5% CO₂ for 45 minutes to solidify the Matrigel, and 1 mL of pre-warmed Mammocult was added to the center of each well. Plates were imaged daily using a Celigo S Imaging Cell Cytometer (Nexcelom) in brightfield mode.

HCT116 and HT29 cells were cultured in McCoy’s 5A medium (Gibco), and DLD1 and COLO320DM cells were cultured in RPMI 1640 (Gibco). All cells were tested to be mycoplasma free. All media were supplemented with 10% FBS, 1% glutamine, and 100 U/mL penicillin/streptomycin. Cells were maintained at 37 °C and 5% CO₂.
For proliferation assays, 5000–7500 cells were seeded in 24-well plates in multiple replicates. Cell proliferation was assessed by measuring confluence using a Celigo S Cell Imaging Cytometer (Nexcelom) every 24 h for 5 days, and plotted as relative confluence over time. The confluence in each well on each day was normalized with the confluence in that well on day one.
For treatment with trametinib (Biomol GmbH), 20 nM of inhibitor was prepared in DMSO and diluted in media. As control, cells were treated with DMSO at a 1:1000 dilution.

Characterization assays were performed as previously described [15 (link)]. Briefly, cells were seeded 24 h post siRNA transfection. Proliferation was tested by seeding 2000–5000 cells onto 96-well assay plates (Corning Life Sciences) and measuring the confluence daily using a Celigo® S cell imaging cytometer (Nexcelom Bioscience LLC). Clonogenic growth was assessed by seeding 500 cells onto a 6-well plate and waiting until macroscopically visible colonies were formed. Colonies were visualized by fixing cells in 4% PFA in PBS for 20 min, washed with PBS, and stained with 1% crystal violet in 20% ethanol for 20 min. Plates were washed with water and scanned. To test the migration potential, 100,000 HCT116 cells were seeded per PET track-etched membrane cell culture insert (8 μm, BD Bioscience) which was equilibrated with serum-free growth medium for 30 min. After 48 h, the inside of the inserts was scraped with a humidified cotton swab to remove non-migrated cells. Afterwards, migrated cells were fixed in 100% methanol for 10 min, stained with 0.1% crystal violet in 20% ethanol for 20 min, and rinsed with water. The stained area (%) covered by cells was quantified using FIJI [17 (link)].