Jan 16, 2024
Version 1
Lentivirus Production V.1
This protocol is a draft, published without a DOI.
- 1University of California, San Diego
Protocol Citation: Yiqin Shen 2024. Lentivirus Production. protocols.io https://dx.doi.org/
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: January 16, 2024
Last Modified: January 16, 2024
Protocol Integer ID: 93616
Keywords: lentivirus through cell transfection, lentivirus production, lentivirus, cell transfection
Abstract
A protocol to produce lentivirus through cell transfection
Day 1
Coat 15ml cell culture plates with D-poly-lysine (50ug/ml) for 00:30:00 to01:00:00 .
Wash plates with cell culture water for 1 to 3 times. Let the plates to air dry.
Plate pre-grown low passage HEK cells at 6e^6 cells per plate.
Add 20ml of media.
Incubate overnight at 37 °C 5% CO2.
Note
Cell culture media: DMEM/F-12, GlutaMAX‱ supplement + 10% FBS + 1% P/S
Day 2
Change media about 3 hours prior to transfection.
Transfect cells with viral components using CaCl2 transfection Kit in the afternoon.
Each plate = 20/25ug desired plasmid, 10.2ul PMD26, 15.6ul PsPAX, 93 ul CaCl2; add water to 750 ul; 750 ul HBSS
Add HBSS to tube 2; add plasmid, CaCl2, H2O to tube 1, and mix by vortex.
Add tube 1 to tube 2 (on medium speed vortex) drop by drop (to form droplets).
Wait 00:30:00 ; vortex again before use.
Add 1.5 mL to each plate, distribute evenly across the plate.
Rock the plates gently to allow more distribution.
30m
Incubate overnight at 37 °C 5% CO2.
Day 3
Change media for all of the plates. Add 20 mL fresh media.
Day 4
Collect media from the plates (~24 hours after media change). Store at 4 degrees.
Add 20 mL of fresh media.
Day 5 & 6
5h 25m
Repeat collection as day 4.
Spin down collected media at 500 x g, 4°C, 00:10:00 .
10m
Add 36ml of supernatant from step 10 to 12 ml of Lenti-X concentrator. Incubate the mixture at 4 °C in a rocker, for 04:00:00 to Overnight .
4h 30m
Centrifuge the mixture from step 11 at 1500 x g, 4°C, 00:45:00 . After centrifugation, an off-white pellet will be visible.
45m
Remove supernatant from the tubes.
Resuspend the pellet in 100ul HBSS per pellet.
Aliquote 200ul per freezing tube.
Place the tubes on ice or freezing blocks immediately when you are finished, and transfer to -80 °C for long term storage.