Hcs studio cell analysis software

For tumor cell phagocytosis, apoptosis was induced in EO771 cells using 50 μmol etoposide (Abcam) for 16 hours at 37°C, 5% CO₂. Cells were pelleted, washed twice with PBS, and labeled with pHrodo Red succinimidyl ester (Thermo Scientific) per manufacturer’s instructions. Apoptotic EO771 cells were then co-cultured for 1 hour in triplicate at 10:1 in 96-well plates with adherent Raw.shCtl, RawNRP2a^KD, or RawNRP2b^KD cells labeled with Hoechst 33342 solution at 0.1 μg/ml for 10 minutes (Thermo Scientific). Subsequently, unphagocytosed cells were removed by washing with PBS and co-cultures were imaged with the CellInsight CX7 LZR High-Content Screening Platform at 30 minutes and 4 hours and data analyzed using the HCS Studio Cell Analysis Software (Thermo Scientific). Phagocytosis and subsequent processing of pHrodo-labeled EO771 cells in the acidic environment of the lysosome induces red fluorescence with co-expression of red fluorescence with DAPI, reported by the CircSpotTotalArea metric, indicating processing of pHrodo⁺ EO771 cells.

Caspase 3/7 positive cells were determined with CellEvent Caspase‐3/7 Green Detection Reagent following manufacturer’s instructions. Briefly, primary neurons were prepared as for LDH assays and treated with reagent diluted at 8 µM in 5% FBS NBM. Positive control wells were treated with apoptotic inducer staurosporine at 0.1 and 1 µM for 6 h. Cells were fixed in 4% (w/v) PFA and nuclei were counterstained with DAPI. Images were acquired with CellInsight CX7 (Thermo Scientific) using a 10x objective and analyzed with HCS Studio Cell Analysis Software (nuclear segmentation and casp3/7 intensities). Quantification of percentage of caspase‐3/7 positive cells was done for each well by applying a threshold manually, based on nuclear segmentation and across 81 fields.

YEPD broth was inoculated with C. albicans strain GP100 (expressing the GFP-YPT72 fusion and cytoplasmic mCherry) and grown overnight at 30°C, 180 rpm. Following 1:200 dilution in YNB medium, test compounds were added to a final concentration of either 5 or 25 μM, and 100 μL of each cell suspension seeded to the wells of a U-bottom 96-well plate (Corning #3799). Cultures treated with an equivalent amount of DMSO solvent (0.5%), or 5 μM fluconazole provided negative and positive controls of vacuolar disruption respectively. Following overnight incubation at 30°C plate cultures were resuspended and diluted 1:18 with fresh YNB medium, before 50 μL was transferred to a 96-well glass bottom assay plate (Corning #4580). Assay plates were spun at 400-500g for 5 min at room temperature, then stored in the dark for 1 hour to allow yeast to settle to bottom of the wells. Images were acquired and analyzed on a Thermo Fisher Cellomics Array Scan VTI HCS Reader using HCS Studio^™ Cell Analysis Software. The filter settings for each dye were BGRFR 549–15 for yeast cytoplasm (mCherry-Channel 1) and BGRFR 485–20 for yeast vacuole (GFP-Channel 2). Images were acquired using a 40X objective for a total of 16 different fields in each well. The Spot Detector BioApplication was used to identify the cells in Channel 1 and to identify and measure the yeast vacuole size and number in Channel 2.

Primary cortical neurons grown on poly‐L‐lysine hydrobromide‐coated glass coverslips were fixed in 4% (w/v) paraformaldehyde (PFA) in 1xPBS at room temperature (RT) for 20 min, permeabilized with 1X PBS and 0.2% Triton X‐100 for 10 min at RT, and blocked for 1 h in 1x PBS and 0.1% Triton X‐100 with 5% (w/v) BSA. Neurons were incubated with primary antibodies (Appendix Table S3) overnight at 4°C. After three washes in 1× PBS and 0.1% Triton X‐100 for 10 min, neurons were incubated with secondary antibody anti‐rabbit and anti‐mouse (Appendix Table S3) for 1 h at RT, and subsequently washed three times in 1xPBS and 0.1% Triton X‐100 for 10 min. Coverslips were mounted with antifading medium (Vectashield, Vector) with DAPI and analyzed by fluorescence microscopy. Images were acquired with CellInsight CX7 (Thermo Scientific) using a 10× objective and analyzed with HCS Studio Cell Analysis Software (nuclear segmentation, NeuN and GFAP intensities). Quantification of positive cells for each of these staining was done by applying a threshold manually, based on nuclear segmentation and across 81 fields. Percentage of cells positive for NeuN and GFAP staining was quantified for each well.

Cells were seeded into 384 or 96 well #1.5 glass bottom plate (Nunc or Cellvis). Images were captured using EVOS 2 FL (Invitrogen) and analyzed using HCS Studio Cell Analysis Software (ThermoFisher).

For high-content quantitative analysis, images were acquired using an automated microscope (Arrayscan VTI HCS Reader, Thermo Fisher Scientific) of the screening facility. To count the number of hiPPCs expressing mCherry (red) or immunofluorescence-stained cells, an image analysis workflow was created using HCS Studio Cell Analysis Software (Thermo Fisher Scientific) with the TargetActivation BioApplication. Image analysis included an image preprocessing step followed by a segmentation step, allowing classification of the pixels and objects based on fluorescence intensity thresholds. High-content quantitative analysis of hiPPCs derived from hiPSC-2 line were acquired using the automated microscope CQ1 (Confocal Quantitative Image Cytometer, Yokogawa) » and analyze in by the CellpathFinder Software (Yokogawa).