May 29, 2025
Astrocyte Viral Transduction
- Camille Goldman1
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Camille Goldman 2025. Astrocyte Viral Transduction. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjbwxpgk5/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: 218387
Keywords: ASAPCRN, astrocyte viral transduction protocol for transducing astrocyte, astrocyte viral transduction protocol, transducing astrocyte
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 transducing astrocytes with lentivirus
Troubleshooting
Coate desired number of wells of a 6 well plate in 0.1% gelatin
Wash astrocytes with 1x PBS
Dissociate astrocytes in a trypsin enzyme solution (TrypLE Select, Gibco 12563011). For a 10cm plate of astrocytes, use 3mL per well
Transfer dissociated cells to a falcon tube with 10mL astrocyte media (AM; ScienCell #1801)
Centrifuge at 300rcf for 3 minutes at room temperature
Resuspend cells in 1mL of AM and count
Thaw lentivirus on ice
Transfer between 1x105 and 3x105 cells into a 1.5mL microfuge tube, 1 tube per viral transduction
Add lentivirus to the microfuge tube, so that the final concentration once cells are plated with the total volume of media will be 1:50 (40uL of lentivirus when final volume will be 2mL in a well of a 6-well plate)
NOTE: Viral titration may need to be determined on a batch to batch basis, depending on the lentivirus plasmid and the cells being transduced.
Incubate cells in the microfuge tube with the higher lentivirus concentration for 5-10 minutes at room temperature
Add the cell/lentivirus mixture to the prepared well and bring the final volume of AM to 2mL
After 24hrs, change to fresh AM media
If antibiotic selection is necessary, start adding at least 3 days after transduction, and continue selecting for at least 2 days