Hcs studio software

HeLa cells stably expressing the XPO1-dependent NLSSV40-AcGFP-NESPKI reporter were cultured as described previously (41 (link)). To assess the CRM1-mediated nuclear export function, reporter cells were seeded at 8,000 cells per well in 96-well tissue culture plates (Switzerland). The next day, cells were treated with a serial dilution of 4-OI or DMSO for 24 hours, fixated, and counterstained with DAPI. Fluorescence was imaged on an ArrayScan XTI High Content Reader (Thermo Fisher Scientific). Nuclear and cytoplasmic compartments were segmented, and their average pixel intensities in the green channel were quantitated employing the HCS Studio software (Thermo Fisher Scientific). GraphPad Prism was used for dose-response curve fitting based on the percentage of cells having a predominant nuclear localization of the reporter construct. A ratio of nuclear to cytoplasmic signal equal or above 1.4 was considered predominantly nuclear. Representative images were taken by confocal microscopy on a Leica TCS SP5 confocal microscope (Leica Microsystems), employing an HCX PL APO 63× (NA 1.2) water immersion objective.

Cells transfected with lentivirus stably expressing green fluorescent protein (GFP) were seeded into 96-well plates and treated with increasing doses of 5-FU (0, 10, 20 µg/mL for GC cells or 0, 2, 4 µg/mL for GES cells). Cells were imaged every 4 h for 48 h using the Thermo Scientific CellInsight CX7 High Content Analysis Platform (0, 10, 20 µg/mL for GC cells or 0, 2, 4 µg/mL for GES cells; Thermo Fisher Scientific, Cambridge, Massachusetts, USA). Proliferation curves were plotted and analyzed using HCS Studio Software (Thermo Fisher Scientific).

Cells were cultured in 96-well plates (10386612, Fisher Scientific). On day 12 of differentiation, cells were fixed in PBS with 4% paraformaldehyde (SC281692, Santa Cruz Biotechnology) for 10 min and washed with PBS. LDs and nuclei were stained with BODIPY 493/503 (1:2,500 dilution, D3922, Thermo Fisher Scientific) and Hoechst (1:5,000 dilution, ABCAAB228551, VWR) for 10 min. Scanning was performed using CellInsight CX5 (Thermo Fisher Scientific) and quantified by employing object (nuclei) and spot (LD) detection algorithm in HCS Studio software (Thermo Fisher Scientific). LD area and number were normalized to nuclei count.

Recombinant VSV bearing glycoproteins of either ANDV or SNV and expressing green fluorescent protein were incubated with dilutions of vaccinated hamster sera (20, 60, 180, 540, 1620 on Vero cells or 2-fold serial dilutions starting at 100 on HUVEC) for 1 h at room temperature (RT) and then used to infect cell monolayers in duplicate. Cells were scored for infection at 14 h post-infection via GFP expression by automated counting with a CellInsight CX5 fluorescence microscope and onboard HCS Studio software (Thermo Fisher). Percentage of relative infection was determined as compared to infection in the absence of serum. The Reed–Meunch method was used to calculate NT80, the titer of serum at which ≥80% reduction of relative infection is seen.

The survival of all neurons regardless of their neurotransmitter phenotype was monitored by counting MAP-2⁺ cells. Briefly, fluorescent images of cultured cells were acquired with an Eclipse TE-2000 inverted fluorescent microscope (Nikon, Champigny-sur-Marne, France) equipped with an ORCA-ER digital camera (Hamamatsu Photonics, Massy, France) operated with the HCI software (Hamamatsu Corp., Bridgewater, NJ, USA). In each culture well, 10 digitized images randomly acquired with a ×10 objective were taken for cell counting using the image processing program Image J. Counting of ChAT⁺ neurons was performed on digitized images acquired with a ×10 objective mounted onto an Arrayscan XTi automated workstation equipped with the HCS Studio Software (Thermofisher Scientific, Courtaboeuf, France). Mosaic reconstruction of partially overlapping digitized images allowed the counting of ChAT⁺ neurons over >60% of the surface area of each culture well.

Caov-3 cells were seeded into black-walled µClear 96-well plates at a density of 4000 cells per well in 100 µL of complete growth media and allowed to adhere for 24 h at 37 °C with 5% CO₂. The media was removed and replaced with complete media containing 5 µM, 10 µM, 50 µM, or 100 µM artesunate, 0.1% DMSO as a negative control, or 25 μM cisplatin as a positive control. Cells were incubated with drugs for 48 h and then fixed for 15 min at room temperature in 4% paraformaldehyde. We used 0.25% Triton X-100 to permeabilize the cells for 15 min and then blocked in 0.1% bovine serum albumin (BSA) for one hour. We assessed DNA damage with immunofluorescent staining for phosphorylated histone H2AX (pH2AX) using the HCS DNA Damage Kit (Invitrogen). We used the Cell-Insight CX7 High Content Analysis Platform for imaging and HCS Studio software to quantify the nuclear pH2AX signal (both ThermoScientific). We completed the statistical analysis of pH2AX signal on GraphPad Prism (version 5.01).