Lumox 24 multiwell plate

Lung cells and breast cancer cells were plated onto Lumox 24-multiwell plate (Sarstedt, 94.699.00.14) in Mitogen Low-Nutrient Low medium (MLNL, low glucose DMEM/1%FCS, Thermo Fisher Scientific 21885025) or Mitogen High-Nutrient High medium medium (MHNH, high glucose DMEM/10%FCS, Thermo Fisher Scientific, 41965-039) as indicated. In detail: AT1-like cells (12,5x10⁴ cells/well) and AT2-like cells (2,5x10⁴ cells/well) were plated at day 1, HNLFs at day 2 (2,5x10⁴/well) and cancer cells at day 3 (100 cells/well). Medium was replaced every three days and GFP⁺ cells were manually counted under an inverted fluorescent microscope after replacing medium with HBSS. For experiments where relative number of cells/ml is shown, cells from each well were trypsinized, filtered through a 70μm cell strainer and resuspended in 200μl of FACS buffer (PBS, 2mM EDTA, 3%BSA). Number of GFP⁺ cells/ml was then measured with MACSQuant Analyzer (Miltenyi Biotec) with 96well plate module.

2x10⁴ AT1-like cells/well were plated onto Lumox 24-multiwell plate (Sarstedt, 94.699.00.14) in MLNL or MHNH medium as indicated. The following day 2000 D2.0R cells were plated in the same media. 3-4 hours after plating the indicated inhibitors were added and imaging for 48 hours using either a LSM510 or Nikon Eclipse Ti2 was commenced two hours later. The movies were analyzed manually to record the number of cells at the beginning, at the end, the number of mitoses, and the number of cell death events.