Cellcarrier ultra 96 well

Cells were grown on PerkinElmer CellCarrier Ultra 96-well (Waltham, MA, USA) plates until 70% confluency. Samples were fixed with 4% formaldehyde for 15 min, permeabilized with 1% Triton-X 100, and blocked with 1% BSA/PBS solution. Nuclei were labeled with DAPI (Thermo, Cat. No. R3706) for 5 min. Actin filaments were labeled with Texas Red-X phalloidin (Thermo, T7471) for 1 h. Fluorescent images of the nuclei were acquired at a resolution of 1080 × 1080 pixels (px) with a 10× objective (N.A. 0.3, 1.196 μm/px). DAPI signals (excitation/detection λ: 405/456) were detected in the whole well with 21 fields. Morphology images (i.e., fluorescent detection of phalloidin-stained cells) were acquired at 2160 × 2160 px image resolution, with a 20× objective (N.A 0.4, 0.299 μm/px). DAPI and Texas Red-X phalloidin signals (excitation/detection λ: 561/599) were detected in 25 fields from each well. Both image sets were acquired with PerkinElmer Opera Phoenix High Content Screening System (Waltham, MA, USA).

U-2 OS cells stably expressing Sec61β-mEmerald [33 (link)] were seeded at 4100 cells per well (equivalent to 1.3 × 10⁴ cells per cm²) in CellCarrier Ultra96-well optically clear plates (PerkinElmer). 16-h treatments started 24 h after seeding while 30-min treatments started at 39 h 30 min after seeding. Staggered treatments allowed synchronised fixation and staining of the entire plate. Cells were treated for 16 h with 0.5% DMSO (Sigma-Aldrich D2650), 250 μM Palmitic acid (Sigma-Aldrich P0500), 50 nM Bafilomycin A1 (B1793 Sigma-Aldrich), 100 nM Thapsigargin (Sigma-Aldrich T9033), and 500 nM Tunicamycin (Sigma-Aldrich T7765); and for 30 min with 10 μM Cytochalasin B (Sigma-Aldrich C6762).