Incucyte s3

Cells were plated on 96 well plates (Corning Cat. no. 353072) in 200 μL of DMEM supplemented with FBS. Plates were then inserted into the Essen Bioscience Incucyte S3 imaging system with plates imaged every 6 h. After 48 h of growth, cells were treated with inhibitors and pH 7.4/6.5, and imaging continued every 6 h. After seven days from initial plating, Incucyte images were analyzed for confluence using the provided Incucyte S3 software. The confluency was plotted against time, and p value was calculated using the GraphPad Prizm 8.1.1 program. The outer two rows as well as the outer two columns were disregarded from analysis due to edge-related evaporation effects of the plates.

To quantify GFP expression, cells were plated in clear flat-bottomed 96-well plates (Nunc) and imaged every hour using the IncuCyte S3 live cell imaging system (Essen BioScience). Five fields of view were taken per well at 10× magnification, and GFP expression was determined using the total integrated intensity metric included in the IncuCyte S3 software (Essen BioScience). To analyse images generated on the IncyCyte S3, a collection of representative images is first taken to set fluorescence and cellular thresholds, which allows for the removal of background fluorescence, and selection of cell boundaries (‘objects’) by creating ‘masks’. Following this, the total integrated intensity metric can be accurately calculated by the software, which takes the total sum of objects’ fluorescent intensity in the image, expressed as green count units (GCU) µm⁻².

Wound healing of the HGSOC cells was measured for 72 h at 2-h intervals with an IncuCyte S3 high-content imager (Essen Bioscience, Ann Arbor, MI). Experiments were performed on 96-well plates (ImageLock, Essen BioScience) with adavosertib (500 nM) or vehicle (DMSO). Each sample was measured in triplicate, and the experiments were repeated a minimum of three times. Relative wound density was analyzed by IncuCyte software (Essen BioScience). The wells were precoated with Geltrex (Gibco) for migration experiments and with Matrigel (100 µg/ml, Corning, Bedford, MA, USA) for invasion experiments. Wells were pretreated for 24 h with adavosertib (500 nM) before wound making with a wound-maker provided with IncuCyte S3 (Essen Bioscience). In the invasion experiments after wound making, the cells were covered with 50 µl Matrigel (2 mg/ml) for 30 min in the incubator; thereafter, adavosertib was added.

For cell cycle analyses, cells were harvested with trypsin (72 h post-transfection or otherwise indicated), fixed overnight in 70% ethanol at −20°C. DNA was stained with propidium iodide (Miltenyi Biotec; diluted 1:1000) at 37°C for 30 min in PBS supplemented with RNAse A (2 μg/ml; Sigma Aldrich) to deplete RNA. The DNA content was measured by flow cytometry using a MACS Quant Analyzer (Miltenyi Biotec) and analyzed using FlowJo. The FUCCI system was used to analyze the length of cell cycle phases. ES-2 cells, stably transduced with IncuCyte^® Cell Cycle Red/Green Lentivirus Reagent (Sartorius), were transfected with indicated siRNAs. Cells in the G2/M phase were enriched by FACS based on their green fluorescence using a FACS Melody sorter (BD Bioscience) 24 h post-transfection. Cell cycles phases were monitored based on their fluorescence using an IncuCyte S3 (Sartorius) starting immediately after sorting. Cell segmentation and quantification was performed using the Cell-By-Cell module (IncuCyte S3; Sartorius). Single cell tracking was subsequently processed using ImageJ.

The antibody binding and blockade assays were performed as described previously (41 (link)). Briefly, to measure PD-L1 protein and PD-L1 antibody interaction, we seeded 1 × 10⁴ BT549^cPD-L1 cells per well in 96-well plates and then incubated the plates with cIgG control (Rockland Immunochemicals), or 12C10E4 antibody, and anti-canine Alexa Fluor 488 dye conjugate (SouthernBiotech). Every hour, green fluorescent signal was measured and quantified by IncuCyte S3 (Sartorius). To measure PD-1 protein on the cells, we seeded 1 × 10⁴ BT549^cPD-L1 cells per well in 96-well plates, and then incubated the plates with cIgG control (Rockland Immunochemicals), or 12C10E4 antibody, cPD-1-hFc protein (human Fc protein conjugated; SinoBiological US), and/or anti-human Alexa Fluor 488 dye conjugate (Thermo Fisher Scientific). Every 3 hours, green fluorescent signal was measured and quantified by IncuCyte S3 (Sartorius). The Image analysis was performed according to the manufacturer's protocol.