Cytation3 imaging multi mode reader

For the evaluation of cellular responses to both incubation with DON and uniaxial stretching, live cell imaging experiments were performed. Cellular structures/organelles were stained with specific dyes: cell membrane was stained with CellMask™ Deep Red Plasma membrane Stain (1:1000 dilution, white), lysosomes with LysoTracker® Red DND-99 (1:1000 dilution, red, indicated as LysoTracker), mitochondria with MitoTracker® Green FM (1:1000 dilution, green, indicated as MitoTracker), tubulin with TubulinTracker™ Green (1:2000 dilution, green, indicated as TubulinTracker), and cell nuclei with Hoechst 33258 (1:1000 dilution, blue). Staining solutions were diluted in Live Cell Imaging Solution (all from Molecular Probes, Life Technologies, Thermo Fisher Scientific, Waltham, USA). At the end of the staining, cells were rinsed and maintained in Live Cell Imaging Solution for the microscopy analysis. Time series and confocal images were acquired with Confocal LSM Zeiss 710 equipped with ELYRA PS. 1 using a Plan Apochromat 63X/1.4 oil objective (Figs 1–3), whereas membranes were imaged with a Plan Neofluar 10X/0.3 (zoom 4; Figs 4–6). Moreover, membranes were also analyzed with the Cytation3 Imaging Multi-Mode Reader (BioTek, Winooski, VT, USA) for image acquisition and evaluation of the fluorescent signal (objects-mean per optical field; Fig. 7).

Evaluation of membrane fluidity was performed as previously described (Zhang et al. 2011 (link); Del Favero et al. 2021a (link); Rebhahn et al. 2022 (link)). Briefly, cells were seeded in a black 96-well plate with clear bottom and incubated for 24 h with rapamycin 1–100 nM. The day of the measurements, cells were loaded for 1 h with 1-pyrendecanoic acid (10 µM, PDA, Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence emission signals for PDA monomers (375 nm; I_m) and excimers (470 nm; I_e) were measured after excitation (344 nm) with a Cytation3 Imaging Multi-Mode Reader (BioTek, Winooski, VT, USA). Membrane fluidity was expressed as ratio I_e/ I_m and compared to solvent controls. Experiments are mean of 3 independent biological replicates performed at least in technical quadruplicates.

For the performance of the migration assay cells were seeded in Culture-Insert 4 Well in µ-Dish 35 mm, high (Ibidi GmbH, Martinsried, Germany). Cells were allowed to grow to confluency in the wells and incubated for 1 h with 1 µM ATXII or 0.1% DMSO for the controls. At the end of the incubation, toxin-containing medium was removed, cells were rinsed with pre-warmed PBS and the insert was removed and images were acquired to obtain the initial images (time 0). Afterwards, cell type-specific complete medium was replaced. Images were acquired at differential time points and 8 h was chosen for the performance of the quantitative evaluation. Live cell imaging was performed in Live Cell Imaging Solution (Molecular Probes, Life Technologies, Thermo Fisher Scientific, Waltham, USA) using the Cytation3 Imaging Multi-Mode Reader (BioTek, Winooski, VT, USA). Image analysis was performed with Photoshop CC2015, overlapping directly the sequential images and calculating the percentage of area covered by the cells. Data are means ± SE obtained from minimum three independent cell preparations and analyzed with Origin Pro 9.1G (OriginLab, Northampton, USA) applying Mann–Whitney test (threshold values p < 0.05).

Immunofluorescent staining was performed on 4 µm formalin-fixed, paraffin-embedded ovarian tumour sections. After deparaffinization, antigen retrieval was performed by boiling slides in sodium citrate buffer, and permeabilization was performed for nuclear antigens only using 0.25% v/v Triton X-100. Non-specific antibody binding was blocked with 5% BSA in PBS, and primary antibodies were applied and incubated overnight at 4 °C. Primary antibodies used were rabbit anti-wide-spectrum cytokeratin (#ab9377, 1:100, Abcam, Cambridge, UK), rabbit anti-Wilms tumor protein-1 (#ab89901, 1:100, Abcam), goat anti-PD-L1 (#AF1019, 1:125, R&D Systems, Minneapolis, MN, USA) and rabbit anti-p53 (#ab31333, 1:100, Abcam). Alexa Fluor® 594 or 647-conjugated goat anti-rabbit or donkey anti-goat secondary antibodies (1:1000, Abcam) were applied at 1:1000 for 1 h, and slides were mounted using ProLong™ Gold Antifade Mountant with DAPI (ThermoFisher Scientific). Fluorescence images were captured using the Cytation 3 imaging Multi-Mode Reader (BioTek) and processed using the Gen5™ software v 2.05 (BioTek).

The membrane fluidity of HT-29 cells and HCEC was measured according to the protocol described from Zhang and co-workers with slight modifications (Zhang et al. 2011 (link)). Cells were pre-incubated with 1-pyrenedecanoic acid (PDA; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; 37 °C, 1 h) and afterwards stimulated with ATXII or solvent controls. Cholesterol complexing agent methyl-β-cyclodextrin (10 µM, MβCD) was added as comparison. Quantification of the dye in monomeric form was performed at 375 nm and in excimeric form at 470 nm (excitation 344 nm) using the Cytation3 Imaging Multi-Mode Reader (BioTek, Winooski, VT, USA). Data are mean ± SE of minimum four independent experiments performed in technical quadruplicates. Statistical analysis was performed with Origin Pro 9.1G (OriginLab, Northampton, USA) applying one-way ANOVA with Fisher test for pairwise comparison (threshold value p < 0.05).

The levels of the human inflammatory cytokines TNFα and IL-6 and anti-inflammatory factors IL-10 and IL-1Ra produced by cultured monocytes were assessed in cell supernatants by ELISA (R&D Systems, Minneapolis, MN, USA) using a Cytation 3 imaging multi-mode reader (BioTek, Winooski, VT, USA). The presence of IL-1β was not measured because, from preliminary experiments, the production of this inflammatory cytokine in cells challenged with LPS after primary activation and 7 days of resting was below detection even in controls. Each sample was tested in duplicate in ELISA.