Incucyte sx5 live cell analysis system

U87 WT/DN and RADH87 EV/WT/Q* were seeded in 96-well plates at 4000 cells per well and RADH85 EV/WT/Q* at 3000 cells per well and incubated in a 5% CO₂ humidified atmosphere at 37 °C for 24 h. U87 or RADH85 cells were then treated with CD95L (doses and times indicated in the figure legends) in the presence of 250 nM of a fluorescent dead cell marker (Incucyte® Cytotox Red Dye, #4632, Essen BioScience, Germany). RADH87 cells were pre-treated with 200 nM (2X) birinapant during 1 h before adding 1 µg/mL of IgCD95L and Cytotox for 24 h. For cell death assays performed in the presence of MKC-8866, U87 WT were seeded in 96-well plates at 5,000 cells per well incubated in a 5% CO₂ humidified atmosphere at 37 °C for 24 h. Cells were pre-treated with DMSO or MKC-8866 (30 µM) and further treated with CD95L in the presence of 250 nM of Cytotox. Plates were placed in the Incucyte® (SX5 Live-Cell Analysis System, Essen BioScience, Germany). Both phase and fluorescent images were recorded (400 ms exposure, 10xlens). Four images were taken every 2 h. Each condition was recorded in triplicate. Images were analysed using the Adherent Cell-by-cell on Red phase algorithm and Cell-by-cell Classification of High Red Intensity (% of total cells) algorithm from Incucyte® 2022B Rev2 software.

To analyze cell migration, hRPE cells were seeded on ninety-six well plates at a density of 5 x 10⁴ cells per well. A wound was made by scraping hRPE cells monolayer with WoundMaker^TM (Essen BioScience, Inc., Ann Arbor, MI, USA). Cells were treated with either TGFβ2 (10 μg/mL) or with TGFβ2 + αBC-P, and images were analyzed using Incucyte SX5 Live-Cell Analysis System (Essen BioScience, Inc., Ann Arbor, MI, USA) at zero hours, 6, 12, 18, and 24 h after scratch. The percentage of hRPE cell migration was obtained with Image J software (NIH). At least three fields (magnification 4×) of view per treatment of six independent experiments were quantified.

A 1:1 mixture of CVB3-eGFP and either CVB3-mCherry viruses encoding the WT or mutant capsid was used to infect Hela H1 cells at an MOI of 0.001 in a 24-well plate. Automated real-time quantitative fluorescence microscopy was used to track the spread of each virus in an Incucyte SX5 Live-Cell Analysis System (Sartorious). The ratio of the mCherry signal (derived from the WT or mutant virus) to the eGFP signal of the reference virus at 20hpi (Ratio_20hpi) provides a measure of the relative success of both the WT and mutant virus following several rounds of infection. The ratio of mCherry to eGFP signal at 8hpi (Ratio_8hpi) reflects the initial relative abundance of each virus before the competition. To calculate the fitness of each mutant versus that of the WT virus, the formula (Ratio_20hpi^Mutant/Ratio_8hpi^Mutant)/(Ratio_20hpi^WT/ Ratio_8hpi^WT) was used. The results of the competition assay are available on GitHub²³ (section 9).

The viability of human primary macrophages exposed to VLCFAs was investigated using Calcein Red-AM staining. Cells were incubated with C26:0 (10, 20, 50 or 100 µM with final concentration of EtOH filled up to 1%) for 24 h, washed with PBS and stained for 30 min with 1 µM Calcein Red-AM (Biolegend). Plates were imaged using the IncuCyte SX5 live-cell analysis system (Sartorious) with a 10× objective for phase contrast and the orange channel (acquisition time 400 ms). Images were examined using the IncuCyte software. Phase images were subjected to segmentation adjustment of + 1. The analysis definition was optimized for the red channel, so that each red object corresponded to a viable cell by performing a Top-Hat segmentation with a threshold of 15 OCU, a radius of 10 µm, and an edge sensitivity of − 25. By using these settings, red object areas smaller than 80 µm² were filtered out. Data were represented as the number of viable cells per image against phase area per image.

The SARS-CoV-2 HEK293T GFP-split assay was performed as previously described (11 (link)). Briefly, HEK293T-GFP1-10 and HEK293T-GFP11 were mixed at a 1:1 ratio (3 × 10⁵ cells of each cell type per well of a 96-well plate) and transfected with 50 ng of pcDNA3.1-SC2-spike (or an empty vector as a control) and 50 ng of pCG1-ACE2 plasmid using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. Cells were incubated overnight at 33°C and then shifted at different temperatures (33°C, 37°C, or 39°C) for the indicated time (0 h, 3 h, 6 h, or 8 h). Images were acquired in an IncuCyte SX5 Live-Cell Analysis System (Sartorius). The percentage of GFP-positive area and cell confluence was calculated with the IncuCyte analysis software, and the percentage of fusion was calculated as the ratio between GFP confluence and cell confluence.