In cell analyzer 1000

Diploid hPSC sublines (H7.s14, H7.s14-Tomato, or H14.s9) were mixed at different ratios with their aneuploid counterparts (H7.s6-GFP or H14BJ1-GFP) as detailed in Supplemental Experimental Procedures. Ratios of abnormal cells in culture were assessed either by high-content imaging using the InCell Analyzer 1000 (GE Healthcare) and associated image analysis software (Developer Toolbox 1.7, GE Healthcare) or by flow cytometry of a duplicate flask using BD FACSJazz (BD Biosciences). Mosaic cultures were treated with colcemid and processed for G-banding (Supplemental Experimental Procedures).

Purified OPCs were placed on coverslips coated with poly-L-lysine (Sigma) and laminin (Engelbreth-Holm-Swarm murine sarcoma; Sigma; 2 × 10⁴ cells/well) and they were cultured with dual PDE7-GSK3 or GSK3 inhibitors (VP3.15 or TDZD8; 1 μM) in a previously described serum-free differentiation medium78 (link), consisting of BME:F12 (1:1; Invitrogen) supplemented with 100 μg/ml transferring (Sigma), 20 μg/ml putrescine (Sigma), 12.8 ng/ml progesterone (Sigma), 10.4 ng/ml sodium selenite (Sigma), 25 μg/ml insulin (Sigma), 0.8 μg/ml thyroxine (Sigma), 0.6% glucose (Normapur), 6.6 mM glutamine (Invitrogen) and 100 U/mL penicillin, 0.1 mg/ml streptomycin and 0.25 μg/ml Amphotericin B antibiotic anti-mycotic solution (Sigma). After 2 DIV, they were fixed with 4% paraformaldehyde (PFA) and subjected to immunocytochemistry for active caspase 3 (Casp3; 1:200; Abcam; Cat#ab13847) and the OPC marker A2B5 (1:10; Hybridoma Bank). 10–20 microphotographs from each coverslip were taken randomly with an In Cell Analyzer 1000 (GE-HealthCare) and the number of Casp3⁺-A2B5⁺ double positive cells was counted using the software In Cell Analyzer 1000 Workstation (GE-HealthCare). Data were expressed as a mean ratio of double positive cells for Casp3 and A2B5 with respect to the total number of A2B5⁺ cells ± SEM for each condition in at least three independent experiments.

With Hoechst 33342 dye staining and the fluorescence of d2EGFP, a live cell image analysis system-IN Cell Analyzer 1000 system (GE Healthcare, PA, USA) was used to obtain the cell images. The plate was put on the plate holder of an IN Cell Analyzer 1000 system and the environmental control module was adjusted to the same conditions as a CO₂ incubator. The images of CHO cells were acquired every 30 min for 24 h. The plasma membrane spot analysis module was used to determine the level of d2EGFP signal (NF-κB expression) throughout the cell and then identified and quantified the fluorescence intensity of the cell. The activity of NF-κB was represented by the percentage of the cells containing green fluorescence in the counted cells. The NF-κB activation data were collected by the IN Cell Analyzer 1000 every half an hour.

Image acquisition and analysis were performed in 96-well plates. Cells were fixed using 3,7% formaldehyde, and cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (AppliChem, A1001). Cells were kept in PBS, and images were acquired using the IN Cell Analyzer 1000 (GE Healthcare) using the Nikon Plan Fluor ELWD 40 × 0,6 objective. In each well of a 96-well plate, 20 images were taken, each with an average of 10 cells per image. Subsequent automated image analysis was performed using IN Cell Analyzer Workstation 3.4 software^{9 (link),30 (link)}.

Images were captured using an InCell Analyzer 1000 (GE Healthcare). A ×10 objective was used, and four fields of view were captured per well. The InCell Investigator software was used to segment and quantify the images as follows. A top hat algorithm was used to identify cell nuclei (Hoechst stain). Cell boundaries were defined using a multiscale top hat algorithm analysis on the MitoTracker Red CMXRos signal. Parkin puncta were identified using the organelle feature with a multiscale top hat algorithm analysis of the EGFP signal. The output of the InCell Investigator software for each plate consisted of a set of tiff files of the images of the microscope taken at different fields and a xls file with the associated numerical data. The xls file included the data of the individual fields and also the summary data of all of the fields and wells in different sheets. The numerical data were recorded using 84 different parameters as described in File S6. The initial primary screen data set consisted of three replicates of 98 different plates. An additional 78 plates were analyzed after the quality control (QC) stage. The secondary screen data set consisted of six replicates of 12 plates.

OPCs were placed on coverslips coated with poly-L-lysine (Sigma) and laminin (Sigma; 2 × 10⁴ cells/well) and they were incubated with the inhibitors (VP3.15 or TDZD8; 1 μM) in the serum free medium described above. After 42 h in culture, BrdU (50 μM; Sigma) was added for 6 h and after a total of 72 h in culture, the cells were fixed and BrdU incorporation was detected by immunocytochemistry (1:20; G3G4, HybridomaBank) combined with the detection of the oligodendroglial marker Olig2 (1:250; Millipore; Cat#AB9610). After immunostaining, coverslips were examined with an In Cell Analyzer1000 (GE-HealthCare) and 10–20 microphotographs from each one were taken randomly. The number of BrdU⁺-Olig2⁺ double positive cells was counted using the software In Cell Analyzer 1000 Workstation (GE-HealthCare) and the data were expressed as a mean ratio of BrdU⁺-Olig2⁺ double positive cells with respect to the total number of Olig2⁺ cells ± SEM, in at least three independent experiments.

Cells were fixed with 4% para-formaldehyde (10 min at 4°C), quenched with 0.1 M glycine and processed for indirect immunofluorescence. Images were collected by using a widefield microscope (Olympus IX70) coupled to the DeltaVision deconvolution system (GE Healthcare).
For Ki67 assay cells were seeded on 96-well plates (Corning) and stained; images were blindly acquired by InCell Analyzer 1000 (GE Healthcare) and analyzed with the ImageJ software.