Pathway 855 high content bioimager

Fluorescence microscopy images were acquired from 96-well plates (#655090, Greiner Bio-One) using a BD Pathway 855® High-Content Bioimager (Becton Dickinson, Franklin Lakes, NJ, USA) using a protocol previously described^{35 (link)}. Minor adjustments were introduced for HT22 cells related to the microscope exposure time during image acquisition and the threshold to yield a binary (BIN) image during image processing (Fig. S2A). An optimal exposure time of 0.2 s and a threshold value of 60 gray values were empirically determined. All other settings were as previously described. In brief, cells were co-stained with three fluorescent membrane-permeable chemical reporters: TMRM (Tetramethyl rhodamine methyl ester, #T668, Invitrogen), Calcein-AM (#65-0853-39; Thermo Fisher), and Hoechst 33258 (#94406, Invitrogen). TMRM is a cation that accumulates in the mitochondrial matrix according to the magnitude of the MMP (depicted in white) Calcein-AM passively accumulates in the cytosol and upon esterase-mediated cleavage of its AM (acetoxymethyl) ester tail it becomes green fluorescent. Hoechst 33258 binds to the AT base pairs in the minor groove of double-stranded DNA greatly increasing its blue fluorescence signal. Combination of cellular and nuclear descriptors allows for calculation of various ‘derived descriptors’ of which ‘norm cyto area’ reflects a measure of average cell size.

Intracellular collagen content of SMCs and 3T3s in response to PHD2 WT or cko conditioned medium was measured using CNA35-FITC (Kindly provided by prof. Reutelingsperger, Biochemistry department Maastricht). CNA35-FITC shown to bind to collagen type I, III and IV.¹⁸ (link) Samples were analysed using the BD Pathway 855 High Content Bioimager. Data were processed with Attovision and BD Diva software.
SMCs and 3T3s were treated with PHD2 WT or cko conditioned medium for 72 h. Culture medium of SMCs and 3T3s was collected and analysed using Sircol soluble collagen assay as described by the manufacturer (Biocolor, S1000). In comparable subsequent experiments, transcription growth factor (TGF)-β1 was added to the conditioned medium or proteins were heat-inactivated (30 min, 85°C) prior to addition to 3T3 fibroblasts.

As a positive control for apoptosis, the cells were treated with 1 µM Staurosporine (Sigma-Aldrich). For the Annexin-V assay cells were seeded in 96-wells plates and allowed to attach overnight at 37 °C. Cells were treated with CDV (IC₅₀) or PBS for 3 and 6 days. Cells were stained with Hoechst 33,342 (200 μg/mL, Sigma-Aldrich) in culture medium for 15 min at 37 °C. Cells were washed with Annexin-V binding buffer (10 mM HEPES, 140 mM NaCl, 5 mM CaCl₂ in PBS) and stained with Annexin-V-FITC (2.5 μg/mL in Annexin-V binding buffer) for 15 min at 37 °C. Staining intensities of cells were measured in High-Content Imaging. Data was acquired using a BDpathway855 High-Content Bioimager (BD Biosciences). Digitalization and segmentation of acquired data was done with Attovision software (BD Biosciences). Processed data was evaluated by DIVAsoftware (BD Biosciences).

Hippocampus from P0 transgenic and non-tg mice was dissected and dissociated with 0.25% Trypsin/EDTA (Gibco) for 14 min at 37°C. Cells were seeded on 96-well microplate (BD Falcon) pre-coated with 0.5 mg/ml poly-DL-ornithine hydrobromide (Sigma-Aldrich) in cell culture media consisting of Neurobasal-A (Gibco), GlutaMAX-I supplement (Gibco), B27 supplement (Gibco), ßFGF (5 ng/ml, Sigma-Aldrich) and vehicle control (DMSO) (Sigma-Aldrich) or LRRK2-IN-1 diluted in DMSO (0.1μM). Cell culture media was changed every 2 days until neurons were fixed with 3.7% PFA. At day in vitro (DIV) 3, 7 and 14 neuronal cultures were immunostained with mouse anti-ß-Tubulin, Class III antibody conjugated to Alexa Fluor 488 (1:50; BD Pharmingen) and the nuclear marker Hoechst 33342 (1:2000), both diluted in PBS. Fluorescent images were captured using the BD Pathway 855 High-Content Bioimager (BD Bioscience) at 20X magnification and a montage of 25 adjoining images (5x5) per well was obtained. Following image acquisition and using the BD AttoVision V1.6 Software (BD Bioscience) images were processed and analyzed for neurite outgrowth. Several parameters (see Table 1) were measured and then statistically analyzed with GraphPad Prism V6 (two-way ANOVA with Tukey’s post hoc test).