Sean Lauber:Adenovirus infection (in vitro)
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PFU = MOI
PFU = MOI #cells
Revision as of 15:54, 16 July 2013
This procedure is optimized for using 96-well plates (0.32 cm^2) with LLC cells. Find out how many cells are ideal for seeding for the time period you wish to study. In the case of LLC cells, 2000 cells/well is good for 48-72 hours.
1. Seed approriate number cells per well (for LLC this is 2000)
2. Incubate overnight at 37°C/5% CO2.
3. Wash cells with 100 ul of PBS two times.
4. Add the appropriate pfu/cell (MOI) of the virus in 20 ul of PBS on top of the cells (you want the media to just cover the cells to allow for high contact between the cells and virus)
5. Incubate at 37°C for 30 minutes.
6. Add 80 ul of 1.25x media on top of the cells (don't remove the virus) - you want this media to contain 1.25x FBS (for 1 ml of 12.5% FBS from 10% add 975 ul 10% media + 25 ul FBS)
7. Incubate for as long as you need at 37°C/5% CO2 (48-72 hours).
Possible to go from 20-50 pfu as well, although 10 seems to work just fine. Consider doing a dose response (MOI = 1, 10, 50).
PFU = MOI / #cells