Opera phenix high content confocal imaging system

For screening of the multicolour cell pool of 41 HAP1 clones, cells were seeded in 12 96-well imaging plates (PerkinElmer PhenoPlate) at a concentration of 2,500 cells per well. At 56 h after seeding, 100 FOVs (approximately one-third of the entire well area; each FOV has a resolution of 1,080 × 1,080 pixels) were imaged per well with an Opera Phenix high-content confocal imaging system (PerkinElmer) using a ×40 water immersion objective. For the treatment of cells with compounds, medium was added to compound plates for pre-diluting compound stocks, before transferring pre-diluted compounds to imaging plates for a final compound concentration of 10 µM and 0.1% DMSO for the majority of compounds. At 6 h after treatment, the same FOVs in compound-treated wells were imaged again as described above.

To generate a cell pool in which every clone can be identified by computer vision, 41 HAP1 clones were selected from the clone collection and thawed individually, before being mixed together in equal proportions using a Sony SH800 cell sorter. The cell pool was expanded for 4 days and frozen in multiple aliquots of 1 × 10⁶ cells per cryotube that were thawed again for screening applications. Clones were also seeded in separate wells of a 96-well imaging plate to generate training data for building a computational model that can identify clones based on localization patterns and intensities in all channels. Each clone was seeded in two wells that were imaged 24 h and 48 h after seeding with an Opera Phenix high-content confocal imaging system (PerkinElmer) using a ×40 water immersion objective.