In cell analyzer 2000

ROS production was evaluated using 2′,7′-dichlorofluorescein diacetate (DCFDA), as described previously^{40 (link)}. The oxidation of the dye by ROS generates a highly fluorescent compound, 2,7-dichlorofluorescein (DCF) that can be detected at λ_ex = 504 nm and λ_em = 529 nm. Briefly, cells were incubated with DCFDA (5 µM) for 30 minutes, washed with warm PBS/5% FBS, and then imaged in a 37 °C, CO₂ atmosphere with a 20× Nikon objective in a high-throughput automated fluorescence wide-field microscope (IN Cell Analyzer 2000, GE Healthcare, Little Chalfont, UK). The acquired images were then analyzed with Developer Toolbox 1.9.2 (GE Healthcare).
Lipid peroxidation was measured with BODIPY®, following the manufacturer’s instructions (Thermo Fisher Scientific). Upon oxidation by lipid hydroperoxides, BODIPY® changes its maximum fluorescence emission wavelength from 590 nm to 510 nm. Briefly, cells were incubated with BODIPY® (10 µM) for 30 minutes, washed with PBS, and then visualized in a 37 °C, CO₂ atmosphere with a 20× Nikon objective in a high-throughput automated fluorescence wide-field microscope (IN Cell Analyzer 2000, GE Healthcare). The acquired images were analyzed with CellProfiler^TM.

Cells were seeded onto 96- or 48-well plates and incubated for 13 days in the presence or absence of 0.2 μM CIS. Colonies were then fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature and stained with 4′,6-diamidino-2-phenylindole (DAPI) for 20 min. After washing, cells were blocked in 5% BSA and 0.1% Tween 20 before staining with anti-CD44 antibody (156-3C11, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. After washing with phosphate buffered saline (PBS), CF488A-conjugated secondary antibodies (Biotium, Hayward, CA, USA) were applied for 1 hour at room temperature. After washing, wells were filled with PBS. Images were collected on an IN Cell Analyzer 2000 automated microscope and deconvoluted using IN Cell Analyzer 2000 software (GE Healthcare). The deconvoluted image projections were analyzed using Developer (GE Healthcare) to identify colonies.

The Momentum 2.0 automation scheduler moved each assay plate from the automated tissue culture incubator (ThermoFisher C2, 37 °C, 5% CO₂) to the barcode reader, then to the automated microscope (GE IN Cell Analyzer 2000), and back to the tissue culture incubator. Each iteration took ~35 min.
A high-content imager (InCell Analyzer 2000; GE Healthcare) was used to collect 20 s of time-lapse images of parasites under one field of view with a ×10 objective. The 4 megapixel charge-coupled device sensor was binned 4 × 4 and the bright-field/4′,6-diamidino-2-phenylindole channel was set to a 3 ms exposure. The focal plane was offset 40 µm from the bottom of the well to thicken the edge (surface) of the worm. The “sit-and-stare” time-lapse schedule began with a 3.5 s delay to allow time for auto-focusing followed by 30 image acquisitions 0.66 s apart.
Images were then analyzed and segmented as described in Supplementary Information, Extended Methods. Features were extracted from the optimal mask chosen from multiple segmentation attempts for each worm and were stored in a custom MYSQL database for visualization in SchistoView.

For throughput compound screening, human iPSCs were dissociated to single cells with TrypLE Express (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) and were disseminated onto iMatrix‐coated 96‐well plates with StemFit containing 10 μm Y‐27632 (Nacalai Tesque, Kyoto Japan). After 24 h, the culture medium was replaced with fresh StemFit containing compounds for 3 h, and then, iPSCs were infected with SeV carrying the EGFP gene. Multiplicity of infection (MOI) was estimated to 1. After 48 h of incubation, cells were washed twice with PBS and then fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature. 4’6‐Diamidino‐2‐phenylindole (DAPI) (Life Technologies, Waltham, MA. USA) was used to label the nuclei. Cell images were acquired with IN CELL Analyzer 6000 (GE Healthcare, Chicago, IL, USA) in the throughput screening and IN CELL Analyzer 2000 (GE Healthcare) in the dose dependency assay, and the number of EGFP‐positive cells was quantified using IN CELL Developer toolbox software 1.92 (GE Healthcare).

To assess cell viability, the LIVE/DEAD test was used using an InCell Analyzer 2000 automated microscope (GE Healthcare, Chicago, IL, USA). Cells of examined cell line were seeded on the surface of tested materials at constant amount of about 12 × 10⁴ per well in 2 mL of medium suitable for a given cell line and then cultured for 48 h under standard conditions. As a control, a well with no sample was used. Before observations under a fluorescent microscope, the medium was removed and the cells were washed with PBS solution. The samples were then incubated for 15 min at room temperature in a balanced Hank salt solution containing a mixture of fluorescent dyes: Hoechst 33342 (Molecular Probes, Eugene, OR, USA), calcein AM (Santa Cruz Biotechnology, Dallas, TX, USA), and propidium iodide (Molecular Probes).
The obtained images were analyzed using InCell Analyzer (GE Healthcare) software. All cells were divided into two subpopulations, i.e., live (stained with calcein-AM—gives green color) and dead (stained with propidium iodide—red color). The total number of cells was determined based on blue fluorescence of Hoechst 33342.

NHEKs were cultured in 96-well culture plates to 40–60% confluence in the presence or absence of cytochalasin D, NSC23766, or MG132. Cells were labeled with Hoechst 33342 (Thermo Fisher Scientific) and treated with BP IgGs (2 mg/ml), normal IgG (2 mg/ml), ColXVII IgG (12 μg/ml), or normal rabbit IgG (2 mg/ml). Live cell imaging was performed with an In Cell analyzer 2000 (GE Biosciences, Piscataway, NJ, USA) equipped with a ×20 objective. After a 6 h incubation with IgGs, the numbers of adherent cells in a ×20 area (0.57 mm²) were automatically counted every hour with the In Cell Analyzer 1000 Workstation software (GE Biosciences) using the cell count analysis module.