Jul 22, 2023

Public workspaceLentivirus production in HEK293FT with calcium-phosphate method 

This protocol is a draft, published without a DOI.
  • Ulduz S. Afshar1
  • 1Bogazici University
Ulduz S. Afshar
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Protocol Citation: Ulduz S. Afshar 2023. Lentivirus production in HEK293FT with calcium-phosphate method . protocols.io https://protocols.io/view/lentivirus-production-in-hek293ft-with-calcium-pho-cxkuxkww
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: July 21, 2023
Last Modified: July 22, 2023
Protocol Integer ID: 85364
Keywords: lentivirus production in hek293ft, lentivirus production, lentivirus, lentiviral vector, hek293ft cell, packaging plasmid
Abstract
To produce lentivirus, HEK293FT cells are transfected with lentiviral vector and packaging plasmids using thecalcium-phosphate method.
Troubleshooting
Day1
Seed 6 million HEK293FT in a 10cm plate with 10 ml complete medium (DMEM+10%FBS+1% pen-strep+1% MEM-NEAA)
Day 2
Add 25 μl from 10 mM Chloroquine (Final Concentration:25μM) to 9 ml of complete medium. (To avoid lysosomal degradation of the transfected DNA)
While preparing the plasmid mix, keep the plates inside the incubator.
In 2ml tubes, prepare the transfection mixture in the following order:
IngredientsStock solution Conc. / mass
pCMV-VSV-G4μg
psPAX27.5μg
Lentiviral Vector10μg
ddH2Ovariable
Total value: 437.5 μl
2M CaCl2 (ide-cold)62.5μl
Transfection mix for Lentivirus production.

500 μl of ice-cold HBS-HEPES buffer saline is added to the mix in a slow dropwise manner.
The Solution was further mixed by passing the tube on a tube rack for 30-45 seconds. until the mixture is full of bubbles.
After 5-7 minutes at RT the mixture was added to the plate drop by drop and with a circular motion to cover the surface of the plates.
After 6 hours, the medium was changed.
Day 4
The medium was collected 48 hours post-transfection and filtered through 0.45 μm filter
The lentivirus-containing medium was aliquoted and stored at -80°C for later use.