Cells were grown in glass chambers (Thermo Fisher Scientific). One hour before imaging, the medium was changed to prewarmed Leibovitz’s L‐15 medium (Thermo Fisher Scientific) supplemented with 20% FBS and 20 mM HEPES, pH 7.0. Recordings were made in a temperature‐controlled incubator at 37°C, as described previously.11 (link), 17 (link), 18 (link), 19 (link), 20 (link) Z‐series of three sections in 3‐µm increments were captured every 15 minutes. Image stacks were projected. Images were collected with an Olympus IX‐71 inverted microscope controlled by DeltaVision softWoRx (Cytiva) using a ×20 0.75 NA UPlanSApo objective lens (Olympus).
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Real-Time Cell Imaging Protocol
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Variable analysis
independent variables
- Time points at which recordings were made (every 15 minutes)
- Cellular imaging (Z-series of three sections in 3-µm increments)
- Temperature (maintained at 37°C)
- Cell culture medium (Leibovitz's L-15 medium supplemented with 20% FBS and 20 mM HEPES, pH 7.0)
- Cell culture vessel (glass chambers)
- Microscope and imaging system (Olympus IX-71 inverted microscope, DeltaVision softWoRx, 20x 0.75 NA UPlanSApo objective lens)
- No positive or negative controls were explicitly mentioned.
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