Incucyte hd

Growth curves were assessed using an IncuCyte HD instrument (Essen Bioscience) using an algorithm to calculate cell confluency based on inverted microscope imaging. Images of each of the 24 wells in the plates were taken every 6 h for a total period of 78 h.

For apoptosis studies, cells were harvested and washed in cold PBS and then resuspended in binding buffer consisting of 10 mM Hepes, 140 mM NaCl and 2.5 mM CaCl₂ pH 7.4. Cells were stained with Annexin V, Alexa Fluor 488 conjugate (Molecular Probes by Life Technologies, Carlsbad, CA, USA) and propidium iodide. Cell cycle distribution and apoptosis were determined by BD FACSCanto™ flow cytometer (BD Biosciences, San Jose, CA, USA) using BD FACSDiva™ software. For cytotoxicity assay, cells were seeded in a 96-well plate and after 12 h YOYO-1 (Life Technologies) was added to a final concentration of 0.1 μM. Object counting analysis was performed using the cell imaging system IncuCyte HD (Essen BioScience).

Growth curves were assessed using an Incucyte ZOOM or an Incucyte HD instrument (Essen Bioscience) using an algorithm to calculate cell confluency based on microscope imaging of the plates. Images were taken every 2 h for a total period of 4 days.

Cell differentiation cultures were imaged from the time of doxycycline addition. Phase-contrast time-lapse images were taken with the IncuCyte HD (Essen Biosciences) inside the incubator, every 15 min, three areas per well. Time-lapse videos were generated with the Fiji software (http://fiji.sc/Fiji) (Schindelin et al., 2012 (link)) using 10 frames per second. Images were analyzed by CellProfiler software (http://www.cellprofiler.org) (Kamentsky et al., 2011 (link)) to quantify the number of round cells with a customized pipeline written by Christian Tischer from the EMBL Heidelberg Advanced Light Microscopy Facility. For each time point, the average and standard deviation of counts from all three spots were calculated to make graphs with Microsoft Office Excel software. A detailed explanation on how time-lapse photography and image analysis were performed can be found at Bio-protocol (Bergiers et al., 2019 (link))

Cells were imaged with an IncuCyte HD (Essen BioScience, Ann Arbor, MI, USA) every 6-12 h during culturing, recording cell confluency for growth curves. Alternatively, since DIP2C KO cells differed in size, growth curves were generated by collection and counting of cells at set time points. For cell size comparison, cell diameter data was collected from the Cedex cell counter (Roche Innovatis, Switzerland) at eight occasions for a total of >5000 cells/cell line. For colony formation analyses, 400 cells plated in triplicate in 6-well plates were stained with 5% methylene blue in methanol after 10 days and colonies quantified. The plating efficiency was calculated as the number of obtained colonies divided by the number of seeded cells. For cell cycle analysis, equal numbers of cells fixed in ice cold 70% ethanol were stained with FxCycle PI/RNase staining solution (Molecular Probes, Eugene, OR, USA) for 15 min at room temperature, washed once in PBS, and analysed using a FlowSight flow cytometer (Amnis, Merck Millipore, Darmstadt, Germany).

To assess wound closure capacity, 10,000 mEPDC were seeded per well of a 96-well plate 16 h prior to scratch initiation, by which point cells have formed a confluent monolayer. A uniform scratch was applied to all wells using the 96-pin WoundMaker (Essen BioScience), and wells were washed twice with media to remove floating cells. Immediately following wounding, cell treatments (12 wells per group) were added to the growth medium and the plate incubated at 37°C, 5% CO₂ until complete wound closure was achieved. Wells were imaged every 4 h using the live content imaging system IncuCyte HD (Essen BioScience) and the integrated analysis algorithm automatically masks each image to identify cell-free and cell-occupied areas in order to calculate relative wound density (RWD; the ratio of the occupied area to the total area of the initial scratched region) for each time point.

NK-cell chemotaxis experiments were performed and analyzed as described elsewhere (Tasdemir et al., 2016 (link)) with the following variations: NK cells were stained with the CellTracker Red CMTPX Dye (Thermo Fisher Scientific) (according to the manufacturer’s instructions) and then seeded into 6-well plates containing proliferating or senescent mVenus-expressing IMR90 cells also expressing the indicated shRNAs, at 60% confluency. Cocultures were imaged over time using an Incucyte-HD or Incucyte-Zoom device (Essen Bioscience) in a 5% CO₂ atmosphere, using a 20x objective and the 488 nm and 561 nm laser excitations. Images were captured every 45 minutes, starting 30 minutes after NK-cell seeding onto the IMR90 cultures. Cell proliferation was determined through repeated-measures of confluency on phase or epifluorescent imaging.