May 29, 2025
Lentiviral Production
- Camille Goldman1
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Camille Goldman 2025. Lentiviral Production. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8ydolmk/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: August 29, 2024
Last Modified: May 29, 2025
Protocol Integer ID: 106634
Keywords: ASAPCRN
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 producing lentivirus
Troubleshooting
Day -1: Preparing HEK Cells
Grow HEK cells in Dulbecco's Modified Eagle Medium (DMEM) with 10% bovine calf serum at 37°C in a 5% CO2 incubator. Use 25 mL for a 15cm plate
Passage ~107 HEK cells per 15cm dish coated in 0.1% gelatin
Day 0: Transfecting HEK Cells
Change media on HEK cells, just prior to the following steps
Combine 14.7 ug pMDLg/pRRE (Addgene #12251), 5.7 ug pRSV.Rev (Addgene #12253), and 7.9 ug of pMD2.G (Addgene #12259) per 15 cm plate
Add 22.5 ug of transfer vector per 15 cm plate
Add 1 mL of 278 uM CaCl2 and mix thoroughly
Add 1mL of 2x BBS solution (280 mM NaCl, 50mM BES, 1.5mM Na2HPO4; pH = 6.95) dropwise. Invert to mix.
Incubate for 1 minute at room temperature
Add dropwise to HEK cells, distributing throughout the plate. Shake the plate on the bench in an X-shape to distribute the plasmid mixture thoroughly
Day 1-3: Collecting Virus
Day 1: After 24 hours, change the media
Day 2: Collect conditioned media after another 24 hours
Day 3: Collect conditioned media after another 24 hours. Combine with the media collected on day 2
Day 3-4: Concentrating Virus
Centrifuge media at 2000xg for 10 minutes to pellet debris
Filter media through a 0.22um PES filter
Add PEG8000 to a final concentration of 5%
Add NaCl to a final concentration of 0.15M
Invert multiple times to mix well and incubate over night at 4°C
Centrifuge at 3000xg for 20 min to pellet viral particles
Discard the supernatant
Resuspend the pellet in DMEM/F12 (or media of your choice) to 1% of the original supernatant volume
Store 50uL aliquots at -80°C