Hh17000001

Cells were fixed with 4% PFA for 15 min at room temperature. Samples were permeabilized and blocked in a single step with 0.2% Triton‐X and 5% donkey serum (Jackson Immunoresearch, 017‐000‐121). Primary antibodies were subsequently added and incubated overnight at 4 °C. Next, cells were washed with PBS, followed by a 40 min incubation with the secondary antibodies. After 10 min of DAPI exposure, cells were imaged using the High Throughput Imager Operetta CLS system (Perkin Elmer, HH16000000). Image analysis and quantification were performed using the Harmony 4.2 software (Perkin Elmer, HH17000001).

End point growth rate experiments of HCT116, HT29, and Caco2 were carried out in 96-well CellCarrier plates (PerkinElmer No. 6005558, Waltham, MA, USA) at an initial cell seeding of 1,500, 4,000, and 2,000 cells per well, respectively. One day after seeding, cells were treated with the indicated dilutions of oxaliplatin (Selleck Chemicals No. S1224, Houston, TX, USA). At the stated time points, images were acquired on an Operetta HCS System (PerkinElmer No. HH12000000) equipped with environmental controls (37°C, 5% CO₂). Thirty minutes prior to imaging, cells were stained with 5 μg/mL of Hoechst 33342 (Invitrogen No. H21492, Carlsbad, CA, USA) and 5 μg/mL of propidium iodine (Invitrogen No. P1304MP, Carlsbad, CA, USA) to determine live or dead cells, respectively. For live cell experiments, cells were seeded on 0.2 or 2 kPa softwell (Matrigen) or CellCarrier plates in the presence or absence of liver ECM disc. Images were taken on the Operetta HCS in confocal mode using the z-stack function. For all experiments, image analysis was performed using the Harmony 3.5.2 software (PerkinElmer No. HH17000001, Waltham, MA, USA). Cells were identified and segmented at the nuclear level to determine live and dead cell counts over time as described previously [32 (link)].