Operetta high content screening system

A compound library consisted of 1280 FDA-approved drugs were purchased from MicroSource Discovery Systems, Inc. (Gaylordsville, CT, USA) [53 (link)]. The source plates containing compounds were used to deliver 10 μM (final concentration) of each drug to 384-well screening plates using an acoustic dispensing Echo 550 instrument (Labcyte, Sunnyvale, CA, USA) and at once the iSLK.219 cells treated by inducers or not were added to these screening plates. The viral supernatants were collected at 48 h post-induction to infect naïve HEK293T cells seeded in 384-well plates by spinoculation as reported previously [31 (link)]. The supernatants were then removed and replaced with fresh medium. At 48 h, fluorescence expression per well was detected using the Operetta High-Content Screening (HCS) System (PerkinElmer, Waltham, MA). Nine image fields per well were recorded by the automated microscope-based HCS and the associated fluorescence intensity per well was calculated using the Harmony 3.5 software (PerkinElmer, Waltham, MA) [31 (link)]. Data were normalized as the fold change compared to the DMSO control. The 50% inhibitory concentration (IC₅₀) for each compound was calculated from these dose-response curves using Graphpad5.0 Prism.

Images were acquired on an Operetta High Content Screening (HCS) System (Perkin Elmer) equipped with climate control (37 °C, 5% CO₂) using a 10X objective lens. Each condition was assayed in at least triplicate wells and a minimum of 24 fields per well per time point (0, 48, 72 hours post drug addition) were imaged. Additional details regarding the imaging protocols used for each cell line can be found in Supplementary Tables 2 and 3.

The clonogenic assay was performed with the Operetta High-Content Screening (HCS) System (PerkinElmer, Waltham, MA, USA) and the surviving fractions obtained as previously described (16 (link), 17 (link)). Briefly, colony assays were performed by seeding cells in 6-well plates at a low density (2,000 cells/well) and allowing growth for 10 days. Colonies were fixed and incubated with 0.05% crystal violet diluted in 20% ethanol for 30 min at room temperature. They were then quantified with the Operetta HCS System (Perkin-Elmer) and the surviving fraction obtained normalizing the counted colonies over the total plated cells, which was expressed as the percentage of control assumed as 100%. Each experiment was performed in quadruplicate.

Based on the results obtained above in the MTS assay, we evaluated the proliferation/migration capacity of fibroblasts using the wound healing assay. For this, canine fibroblasts were seeded at a density of 50,000 cells/cm² in 24-well plates and kept in supplemented medium for 48 hours until they reached confluence. Using a pipette tip, a wound was generated in a straight line in the center of the well. Then, 500 μL of supplemented medium (C-) and the secretome from cLSCs (n = 4) were added.
The cultures were photographed with digital phase-contrast technology in an automated manner using an Operetta High Content Screening (HCS) system (Perkin Elmer) 0, 3, 7, 24, 48, and 120 hours after the application of the treatment.
The size of the open areas (areas without cells, corresponding to the scratch) was calculated by processing and analyzing the images with ImageJ software, applying the MRI wound healing tool (http://dev.mri.cnrs.fr/projects/imagejmacros/wiki/Wound_Healing_Tool). The open areas corresponding to time 0 were considered as 100%, and the rest of the areas were compared to this value for each replicate separately. Additionally, the shrinkage rate of the wound area was calculated for the period between 0 and 48 hours by calculating the slope in terms of mm²/day (Fig 6B). The average ± SD of each condition is shown (n = 4).

To validate the primary cells, cells were fixed with 4% paraformaldehyde (PFA; Sigma, St Louis, MO, USA) for 10 min at room temperature, permeabilized with 0.1% Triton X-100 (Sigma) in Dulbecco’s phosphate-buffered saline (DPBS; Welgene) for 30 min at room temperature, and then washed three times with DPBS. The following primary antibodies were used: mouse monoclonal anti-human serum albumin (ALB; 15C7, ab10241, 1:500; Abcam, Cambridge, UK), mouse monoclonal anti-human Hep Par-1 (Clone OCH1E5, M7158, 1:100; Agilent Technologies, Santa Clara, CA, USA), and mouse monoclonal anti-human CD133/1 (AC133, 130-090-422, 1:100; Miltenyi Biotec, Bergisch Gladbach, Germany). Samples were incubated with the primary antibodies for 16 h at 4 °C and then washed for 10 min three times with DPBS. The secondary antibodies used for staining were goat anti-mouse IgG conjugated with Alexa® Fluor 488 and goat anti-rabbit IgG conjugated with Alexa® Fluor 488 (Invitrogen, Eugene, OR, USA). Samples were then incubated with secondary antibodies for 1 h at room temperature in the dark and washed for 10 min five times with DPBS. For nuclei staining, cells were incubated with Hoechst 33,342 (Invitrogen) for 10 min at room temperature in the dark and washed with PBS twice quickly. All fluorescence images were obtained using the Operetta® High Content Screening (HCS) System (Perkin Elmer, Waltham, MA, USA).

We seeded 2 × 10³ Huh7, Hep3B, or SNU475 cells in each well of 384-well plates (Greiner Bio-One, Monroe, NC, USA). We incubated various combinations of fractions and HO-1089 (H1–H13) for 48 h. The total concentration of HO-1089 and the cell fractions was 50 mg/mL (2-fold dilution, 20-points) in water (v/v). After treating cells with HO-1089 for 48 h, we fixed them at room temperature for 10 min in 4% paraformaldehyde (Sigma-Aldrich), washed them twice with DPBS, and stained nuclei using Hoechst 33342 (Invitrogen, Eugene, OR, USA) for 10 min at R.T. with 20 µg/ml. For every well, we analyzed at least 1000 cells over five microscopic fields, starting at the center. Image analysis was performed with Operetta High Content Screening (HCS) system (Perkin Elmer, Waltham, MA, USA) and Harmony software (Perkin Elmer). We determined the cell counts for the experimental samples and normalized them to the control cells for the relative survival.

Cells were seeded in four 384-well plates 24-h prior to treating with cetuximab (USC pharmacy, Los Angeles, CA, USA). On day 0, cells were treated with the drug at the desired concentration. Before imaging, cells were stained with 5 μg/mL Hoechst 33342 (nuclear dye) (Life Technologies, Grand Island, NY, USA) and 5 μg/mL propidium iodide (PI) (Life Technologies, Grand Island, NY, USA) to identify cells as live or dead, respectively. Individual plates were imaged on days 0, 2, 3, and 5 using the Operetta High Content Screening (HCS) system (PerkinElmer, Waltham, MA, USA)). Cells were then segmented based upon the nuclear dye using Harmony software (PerkinElmer, Waltham, MA, USA). In order to differentiate cell types in co-culture assays, morphological features were calculated and used to train a machine-learning algorithm to classify cells as either ‘CAF’ or ‘tumor,’ as described in Garvey et al. [13 (link),14 (link)]. Propidium iodide intensity levels were calculated and cells were classified as ‘dead’ if their intensity was above the established threshold. Growth rates for each cell type were calculated as previously described [13 (link),32 (link)] by fitting the live cell counts over time to an exponential growth model.