Imagexpress micro xls widefield high content analysis system

To study cell fate during and after mitosis of cells with more than two centrosomes, Pik3ca^H1047R+neo;Flpe-ER^T2 mice were crossed to GFP-Cent2 transgenic mice^{57 (link)} to generate Pik3ca^H1047R+neo;Flpe-ER^T2;GFP-Cent2 MEFs. Cells were seeded in µ-Plate 96 wells (Ibidi; IB-8962) in DMEM medium 48 h after treatment with 4-OHT, followed by filming of the cells for 30 h at 37 °C. To study the efficiency of the G1 checkpoint, an assay of DCB-induced tetraploidy was used. Briefly, MEFs cells were seeded on ibidi µ-Plate 96 wells in DMEM medium with 4-OHT for 24 h, followed by overnight incubation in the presence of 10 µM DCB followed by washing out of this agent in fresh culture and by filming of the cells for 30 h at 37 °C. Images were collected with a high-content analysis microscope (ImageXpress Micro XLS Widefield High-Content Analysis System/Molecular Devices) every 15 min using MetaXpress High-Content Image Acquisition and Analysis Software (Molecular Devices). Time-lapse images from the whole duration of the experiment were then converted into a movie using Image J, and cells with more than two centrosomes or binucleated cells entering mitosis were tracked.

For all experiments, unless otherwise indicated, the ImageXpress Micro XLS Widefield High-Content Analysis System (Molecular Devices^®, San Jose, CA, USA) was used to image cells. The images were analyzed using the applicable modules of the MetaXpress^® High-Content Image Acquisition & Analysis Software supplied by Molecular Devices^® (San Jose, CA, USA).

The indicated cell lines were seeded in appropriate densities in sterile 96‐well plates (Greiner Bio‐One, Kremsmünster, Österreich) one day prior to stimulation with the indicated concentrations of zVAD.fmk, BV6, and human recombinant TNFα. Cell death was assessed by fluorescence‐based microscopic quantification of the fraction of PI‐positive cells, compared to total cells, using Hoechst 33342 and PI double staining (Sigma‐Aldrich). Imaging and quantification were performed using the ImageXpress Micro XLS Widefield High‐Content Analysis System and MetaXpress Software according to the manufacturer's instructions (Molecular Devices Sunnyvale, CA, USA).

For immunofluorescence staining of LC3B, MZ-54 cells were seeded at 10,000 cells/96-well. For immunofluorescence, cells were fixed with 3.7% paraformaldehyde for 10 min, followed by a washing step with PBS and permeabilization with 0.1% Triton-X diluted in PBS for 10 min. After washing with PBS, cells were blocked with an antibody dilution buffer (ADB) containing 0.9% NaCl, 10 mM Tris HCl pH 7.5, 5 mM EDTA and 1 mg/mL BSA for 10 min. Cells were incubated with an antibody against LC3B (Thermo Fisher, PA1-46286) diluted 1:350 in ADB for 1 h. After three washing steps with 0.1% Tween-20 diluted in PBS (PBS-T), cells were incubated with Cy3^TM AffiniPure donkey-a-rabbit IgG (Jackson Immuno Research Laboratories, Inc.) diluted 1:800 in ADB for 30 min. After three washing steps with PBS-T, Hoechst 33342 was added to the cells diluted 1:15,000 in PBS followed by image acquisition with the ImageXpress Micro XLS Widefield High-Content Analysis System (Molecular Devices Sunnyvale, CA, USA) by using the 60× objective and the DAPI and TRITC filter system for acquisition of Hoechst-stained nuclei and Cy3^TM-stained LC3B, respectively. Image analysis was performed using ImageJ 1.51t.

High-throughput microscopy following antiviral phenotypic screening, viral infectivity assay, or end-point dilution assay was performed, as previously described [10 (link)]. In brief, the cells were incubated with virus-specific primary antibody (see antibody details in Table S2) in 0.2% bovine serum albumin (BSA, Sigma–Aldrich, St. Louis, MO, USA) 0.1% Triton-X in PBS overnight at 4 °C. The cells were washed 3× with PBS and then incubated with secondary antibody (Donkey-anti-mouse Alexa-488; 1:800) and DAPI (1:1000, see details in Table S3) in 0.2% BSA 0.1% Triton-X in PBS for 1 h. The cells were washed 3× with PBS and retained in 100 μL PBS for image acquisition. The images were automatically captured with ImageXpress Micro XLS Widefield High-Content Analysis System (Molecular Devices) using 10× Plan Fluor 0.3 NA objective. The images were quantitatively analyzed by open-source software CellProfiler (Broad Institute, Cambridge, MA, USA) pipeline.

Target cells (K562-pCasper cells) were embedded into 2 mg/mL of pre-chilled neutralized bovine type I collagen solution (Advanced Biomatrix) in a 96-well plate. The collagen was solidified at 37 °C with 5% CO₂ for 40 min, after which NK cells were added to the top of the collagen as effector cells. The cells were visualized using ImageXpress Micro XLS Widefield High-Content Analysis System (Molecular Devices) at 37 °C with 5% CO₂. For the 3D killing assay, as described previously [47 (link)], killing events were visualized every 20 min for 36 h, and live target cell numbers were normalized to hour 0 based on area. For live cell imaging to determine time required for killing and the average kills per NK cell, the cells were visualized every 70 s for 14 h and tracked manually. ImageJ software was used to process and analyze the images.

For immunofluorescence staining of LC3B, cells were seeded at 7000 cells/96 well. For immunofluorescence, cells were fixed with 3.7% paraformaldehyde for ten minutes, followed by a washing step with PBS and permeabilization with 0.1% Triton-X diluted in PBS for ten minutes. After washing with PBS, cells were blocked with an antibody dilution buffer (ADB) containing 0.9% NaCl, 10 mM Tris HCl pH 7.5, 5 mM ethylene-diamine-tetra-acetic acid (EDTA) and 1 mg/mL BSA for ten minutes. Cells were incubated with an antibody against LC3B (Thermo Fisher, PA1-46286) or translocase of outer mitochondrial membrane 20 (TOMM20) (Santa Cruz, sc-11415) diluted 1:350 or 1:500, respectively, in ADB for one hour at room temperature. After three washing steps with 0.1% Tween-20 diluted in PBS (PBS-T), cells were incubated with fluorescein (FITC) AffiniPure donkey-anti-rabbit IgG (Jackson Immuno Research Laboratories, Inc.) diluted 1:800 in ADB for 30 minutes. After three washing steps with PBS-T, Hoechst 33342 was added to the cells diluted 1:15000 in PBS followed by image acquisition with the ImageXpress Micro XLS Widefield High-Content Analysis System (Molecular Devices Sunnyvale, CA, USA) by using the 60x objective and the DAPI and FITC filter system for acquisition of Hoechst-stained nuclei and FITC-stained LC3B or TOMM20, respectively. Image analysis was performed with ImageJ 1.51t.