May 29, 2025

Public workspaceLive Cell Imaging

DOI
https://dx.doi.org/10.17504/protocols.io.36wgqqz4ygk5/v1
  • Camille Goldman1
  • 1Icahn School of Medicine at Mount Sinai
Camille Goldman
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DOI: https://dx.doi.org/10.17504/protocols.io.36wgqqz4ygk5/v1
Protocol Citation: Camille Goldman 2025. Live Cell Imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqqz4ygk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 16, 2025
Last Modified: May 29, 2025
Protocol Integer ID: 218390
Keywords: ASAPCRN, preparing cell, cell, cell imaging, imaging
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-024297
NASA
Grant ID: 80ARC022CA004
NIH/NINDS
Grant ID: R01NS114239
NIH/NINDS
Grant ID: UH3NS115064
NIH/NIA
Grant ID: T32AG04968
NIH/NINDS
Grant ID: F31NS13090
New York State Department of Health
Grant ID: NYSTEM-C32561GG
Abstract
Protocol for preparing cells for live-cell imaging
Troubleshooting
Preparing Astrocytes
Refer to the following protocols for astrocyte lentiviral transduction:
https://protocols.io/view/astrocyte-viral-transduction-gzhtbx36p
Plating Astrocytes
No fewer than 3 days after lentiviral transduction, and after completion of antibiotic selection, passage astrocytes into plate for imaging
Coat desired wells of a 96-well, black plate with cover-slip thickness plastic (Greiner 655096 or similar) with 0.1% gelatin
Wash astrocytes with 1x PBS
Dissociate astrocytes in a trypsin enzyme solution (TrypLE Select, Gibco 12563011). For a 6-well plate of astrocytes, use 1mL per well
Transfer dissociated cells to a falcon tube with 5mL astrocyte media (AM; ScienCell #1801)
Centrifuge at 300rcf for 3 minutes at room temperature
Resuspend the cell pellet in 1mL AM and count cells
Plate 1 x 104 cells per well of the precoated 96-well plate
Imaging Astrocytes
Approximately 3 days later, incubate the cells with a live-cell compatible nuclear dye (such as Hoechst33342 or DRAQ5) for 10 minutes
Wash the cells 1x in PBS and replace with fresh AM media
Acquire images on a microscope equipped with an incubation unit and maintain the temperature at 37°C, CO2 at 5%, and humidity at 60%