Live-cell imaging of cell signaling

Cells were plated in a glass-bottomed 24-well plate (CellVis) at ∼5000 cells per well, and grown in Roswell Park Memorial Institute, 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 25 ng/ml amphotericin B for 48 h and then rinsed with PBS and given DMEM Fluorobrite (ThermoFisher) medium with 5% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 25 ng/ml amphotericin B, and 1× Glutamax (ThermoFisher). Cells were imaged every 20 min for 48–125 h by a Nikon Eclipse Ti microscope. The Nikon Perfect focus system was used to maintain focus. An OKO labs incubation system was used to maintain humidity, 37°C, and 5% CO₂. H2B-CFP was imaged using the C-FL AT ECFP/Cerulean Filter Set (Chroma) for 20–40 ms. FOXO1–mVenus was imaged using (Chroma) ET-EYFP Filter Set for 600 ms. HyPer3 probe was imaged using C-FL AT EGFP/FITC/CY2/Alexa Fluor 488 Filter Set (Chroma) for 300 ms. Fluorescence imaging used the Lumencor SOLA light engine. Images were taken using the Hamamatsu ORCA-Flash 4.0 camera with 16-bit depth and 2 × 2 binning. Movies were analyzed using single cell tracking software in MATLAB (Reyes et al., 2018 (link)).