Nov 15, 2022
Spinfection for Suspension and Adherent Cells
- 1University of California, San Francisco
Protocol Citation: Kevin Wilkins 2022. Spinfection for Suspension and Adherent Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2722xg1y/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: November 15, 2022
Last Modified: November 15, 2022
Protocol Integer ID: 72743
Abstract
This protocol is for infection of adherent cells (such as HEK293t) with prepackaged lentivirus.
Different cell lines will have different infection rates, and the infectivity of lentivirus stocks will also depend and vary. Optimization will be required to obtain infection, as well as to determine the appropriate proportion of cells and virus to obtained the desired MOI>
Guidelines
When using lentivirus, adhere to the proper safety measures and precautions. Proper care should be taken that all waste is disposed in biohazard containers or bleached. Double gloves are reccomended.
Materials
Adherent Cells and appropriate media
Lentivirus
Polybrene Polybrene Infection / Transfection ReagentEmd MilliporeCatalog #TR-1003-G
Safety warnings
When using lentivirus, adhere to the proper safety measures and precautions. Proper care should be taken that all waste is disposed in biohazard containers or bleached. Double gloves are reccomended.
Before start
Make and prepackage your virus.
Plating cells
Plating cells
For HEK293t cells, plate 50,000 cells/well in 0.5 mL of DMEM on 24-well plate the day prior.
30m
Spinfection
Spinfection
2h 30m
2h 30m
30+ min before spinfection, make sure the table-top centrifuge is at 30 °C .
30m
Add lentivirus and polybrene to each well.
Virus amount
Virus titer will vary, so it is useful to add different amounts of virus to well to determine the appropriate titer. For a well of a 24-well plate, virus amounts can range from 5 μL to 500 μL.
Polybrene amount
For most cell types polybrene increases infection efficiency and should be used at a final concentration of 8 μg/mL. Example dilution: add 0.8 uL (160 μg) polybrene stock (stock at 100mg/mL) to 0.5 mL media.
Spin at 1000 rcf for 2 hrs at 30 C.
1000 rcf, 30°C
2h
It is OK if the temperature starts off between 20-30C.
Remove plate from centrifuge and place in hood. Douse with ethanol and wait 10 minutes. Place in incubator.
Infection will be achieved within 2-3 days.