Columbus image analysis system

Lysosomal in situ enzyme activity was measured by using lysosome-specific self-quenched substrate (Cat. no. ab234622; Abcam) at manufacturers recommended dosage. In brief, primary mouse cortical neurons were exposed to poly-GA DPRs for 24 h (or were left untreated), and lysosome-specific self-quenched substrate was added during the final 1 h of the 24-h period. Cells were then fixed with 4% PFA at room temperature for 15 min before being imaged with a Zeiss LSM 880 confocal microscope on glass-bottom slides (Ibidi). FIJI was used to analyse the images, and the mean fluorescence intensity of the substrate was quantified per neuronal cell.
Lysosomal staining with the fluorescent acidotropic probe, LysoSensor Green DND-189, was performed according to the manufacturer’s recommendations (#L7535; Thermo Fisher Scientific). Briefly, HeLa cells on a 96-Well Optical-Bottom Plate (#165305; Thermo Fisher Scientific) were exposed to poly-GA DPRs for 24 h (or were left untreated), and LysoSensor Green was added during the final 1 h of the 24-h period (1 μM final concentration). After incubation, live cells were transferred to the PerkinElmer Opera Phenix high-throughput system for imaging (40× 1.1 NA lens). Using the Columbus Image Analysis System, the mean fluorescence intensity of LysoSensor Green was quantified per cell (PerkinElmer).

A total of 10 µL of 0.1 mg/mL Poly-d-Lys solution was added to 384-well plates (Corning #3712) for 5 min, followed by a PBS wash. Plates were dried for at least 2 h; 50 µL of 600000 HEK293 GFP cells/mL of DMEM was dispensed into each well. After 24 h incubation to allow the cells to adhere, 200 nL of small molecules (or DMSO in columns 1, 2, 23 and 24) were pinned. Following a 16-h incubation, 200 nL of 5 mM CCCP was added to all wells except columns 1 and 2, into which 200 nL of DMSO was added. Following 2-h incubation, cells were fixed with 4% paraformaldehyde (PFA) and stained with 50 µL of 1 µg/mL DAPI solution for 15 min. After the final PBS wash, plates were ready for high content microscopy.
Images were acquired on IN Cell Analyzer 6000 (GE Healthcare), equipped with sCMOS camera (2048 × 2048), and 20×/0.45 NA Plan Fluor objective (Nikon) in open aperture mode using 1 × 1 binning. Image analysis was performed using Columbus Image Analysis System (PerkinElmer). Nuclei were initially detected in DAPI channel, followed by whole-cell segmentation in GFP channel. Using PhenoLOGIC machine learning plug-in, cells were categorized into two sub-populations: cells with even GFP-Parkin distribution and cells with GFP-Parkin localized to mitochondria. The percentage of cells with mitochondrial GFP-Parkin was determined per well and averaged across two independent trials.

Cells were treated for 72 h with DAC or AZA at 1 μM and 5 μM concentrations diluted in appropriate medium and imaged and analyzed to evaluate the intensity of EGFP signal as described elsewhere [31 (link)]. The rate of HCT116 cell proliferation (72 h/24 h) in untreated culture types was determined by counting the total number of RFP-H2B-tagged HCT116 cell nuclei using Columbus Image Analysis System (PerkinElmer), as described previously [31 (link)].

For immunocytochemistry and immunohistochemistry, samples were blocked in 10% normal goat serum (NGS) or 10% normal donkey serum (NDS) as appropriate and permeabilised in 0.3% Triton X-100 (Sigma-Aldrich; in PBS) at room temperature (RT) for 1 h. Immunolabelling was performed with primary antibodies in NGS (5%) and Triton X-100 (0.1% in PBS) at 4 °C overnight followed by species-specific secondary antibodies for 1 h at RT and DAPI nuclear counterstain (100 ng/ml) for 10 min at RT. For human post-mortem samples fixation and permeabilisation in cold methanol (−20 °C, 20 min) was performed before the immunostaining. Primary antibodies were diluted as follows: rabbit anti Olig2 (Millipore, AB9610) 1:200; mouse anti SMI32 (Cambridge Bioscience, SMI-32R-500) 1:1000; goat anti ChAT (Millipore, AB144P) 1:100; rabbit anti Pax6 (biolegend, 901301) 1:300; mouse and rabbit anti SFPQ (Abcam, ab11825 and ab38148, respectively) 1:100; mouse and rabbit anti beta-tubulinIII (biolegend, 801201), mouse anti Lim3 (DSHB, 67.4E12) 1:50; chicken anti Nkx2.2 (DSHB, 74.5A5) 1:50. Images were acquired using either a 710 Laser Scanning Confocal Microscope (Zeiss) or the Opera Phenix High-Content Screening System (Perkin Elmer). For image analysis, Fiji or the Columbus Image Analysis System (Perkin Elmer) were used.

For immunocytochemistry, astrocytes were plated onto clear bottom 96 well plates (Falcon). These were fixed with 4% fresh paraformaldehyde for 10 min at room temperature before being washed three times with phosphate-buffered saline (PBS). Samples were then blocked for 1 h at room temperature with 0.3% Triton/PBS/5% goat serum before being incubated overnight with primary antibody in 0.1% Triton/PBS/5% goat serum at 4°C. Primary antibodies used were mouse monoclonal anti-GFAP (Clone G-A-5, 1:500, Sigma-Aldrich) and rabbit anti-cleaved caspase-3 (1:300, Cell Signaling Technology #9661). After three washes in PBS, a secondary antibody (goat anti-mouse, Alexa Fluor 488 or 568, 1:1000) in 0.1% Triton/PBS/5% goat serum was next applied for 1 h at room temperature. Nuclei were counterstained with DAPI (100 ng/mL).
Images were acquired using the automated Opera Phenix High-Content Screening System (Perkin Elmer). For automated image analysis, the Columbus Image Analysis System (Perkin Elmer) was used. Five non-overlapping fields per well were acquired, and two wells per condition (total >5000 cells) were analyzed.

iPSCs were cultured in 6-well plates (Corning) and were differentiated into endothelial cells as described earlier. At day 3 cells were fixed in 4% paraformaldehyde (PFA) at room temperature for 20 min, permeabilized and immunostained following standard protocol using anti-Factor VIII antibody (Abcam, ab236284, 1:200) and fluorescently labeled secondary antibody (Invitrogen, Alexa fluor 546 goat anti-rabbit, 1:500). Additionally, nuclei were labeled with Hoechst 33342 (Invitrogen, H3570, 1:5000). Images of cells were automatically captured using an automated Operetta CLS confocal microscope (PerkinElmer) at 20× magnification. Subsequent image capturing was performed using the PerkinElmer Columbus Image Analysis System as previously described^{63 (link)}.