Incucyte flr

Phase-contrast time-lapse microscopy images were acquired using the IncuCyte FLR (Essen BioScience Inc.) located inside the incubator. The microscope had a 20× objective with the ability to capture high quality phase-contrast microscopy images, 1040×1392 pixels each. Seventeen frames/images per experimental well were acquired, one every 4h. The total size of image data per 384-well plate was 5.6GB (5236 images).

For cell proliferation assays 2^∗10⁴ cells were seeded in 24-well plates and allowed to attach for at least 16 hours. Medium was then changed to normal DMEM or DMEM with added/substituted nutrients or drugs. Cell growth in galactose was performed by culturing cells in glucose-free and pyruvate-free DMEM (Life Technology cat. no. 11966-025), with added 10% v/v FBS, 25 mM D-galactose (Sigma, G0750) and 1 mM sodium pyruvate (Sigma, P2256). To assess cell proliferation in the presence of aminooxiacetate (AOA) and aspartate normal DMEM was supplemented with varying concentrations of AOA (Sigma-Aldrich, C13408) and 4 mM L-aspartic acid (Sigma-Aldrich, A9256). At least 4 independent replicates were recorded for each condition, in each experiment. Cell growth was assessed with an IncuCyte FLR (Essen Bioscience) and assay was stopped when all cell conditions reached full confluency or confluency started to decrease consistently.

6^∗10⁶ cells were seeded in a 96-well plate and cultured overnight in standard conditions. A 700-800 μm wound was obtained in each well with a 96 pins IncuCyte WoundMaker (Essen Bioscience). After applying the wound, cells were washed with PBS twice and medium was replaced with 100 μL DMEM. Images were acquired with an IncuCyte FLR (Essen Bioscience) every 2 hours for at least 10 consecutive hours. Wound widths at time point 0 hours and 6 hours were extracted and used to calculate migration speed. 8-16 wells per condition were used as technical replicates in each experiment, and at least 4 independent experiments were acquired.

Short-term cell death was determined with an Incucyte FLR or Zoom imaging system (Essen Bioscience)⁵⁸ . Cells were treated as indicated in the Figure legend together with 30 nM Sytox green and imaged every 1 or 2 h. Analysis was performed using the Incucyte software and the number of dead (Sytox green positive) cells was normalised to the confluency at t = 0. Alternatively, cells were pre-treated for 1 h with 50 nM Syto 21, followed by cytotoxic treatments as indicated together with 5 μg/ml propidium iodide. Long-term colony formation assay was performed by plating 1000 cells per 6 well and treatment as described in the Figure legend. After 48 h of treatment with supernatant, the medium was changed to 2.5 μM venetoclax/S63845. Medium was changed to regular growth medium after an additional 48 h, and resulting colonies were stained with crystal violet after an additional 5 days. Cell death analysis via FACS was performed using Annexin V - propidium iodide staining^{59 (link)}. In short, treated cells were harvested and stained with 5 μg/ml propidium iodide and Annexin V (Biolegend) in Annexin V-binding buffer (10 mM Hepes pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) for 15 min. Flow cytometry was conducted on a BD FACSCalibur machine with BD CellQuest software and analysed using Flowing software; cells negative for propidium iodide and Annexin V were considered alive.

For a standard BrdU-assay, 10 000 cells were plated in 100 μl of normal growth medium containing vehicle or the indicated bisphosphonates for an indicated length of time. Incorporated BrdU was measured with a commercial kit, according to the manufacturer's recommendations (Exalpha Biologicals, Watertown, MA). For a standard MTS assay, the cells were plated on 96-well plates (10 000 cells per 100 μl per well) in normal growth medium, supplemented with 1-100 μM of the indicated BPs or vehicle as a control treatment. Cell viability was measured with the CellTiter 96 Aqueus One Solution Cell Proliferation assay (Promega, Madison, Wisconsin), according to the manufacturer's recommendations. Cell growth was also measured as a function of confluency, using IncuCyte FLR and IncuCyte ZOOM® (Essen BioScience Ltd., Hertfordshire, UK) kinetic high-content live cell microscopes. Briefly, 2000-4000 cells were plated in 100 μl per well to 96-well plate with the desired concentrations (1-100 μM) of indicated BPs or vehicle. The default software parameters with a 10× objective were used for imaging. The IncuCyte 2010A or 2014A software (Essen BioScience Ltd.) was used to calculate mean confluences of the individual wells (3-8 parallel wells per treatment group) from phase contrast images.