Sean Lauber:Adenovirus infection (in vitro): Difference between revisions
No edit summary |
No edit summary |
||
Line 1: | Line 1: | ||
This procedure is optimized for using 96-well plates (0.32 cm^2). | 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 2000 cells per well | 1. Seed 2000 cells per well | ||
Line 7: | Line 7: | ||
3. Wash cells with 100 ul of PBS two times. | 3. Wash cells with 100 ul of PBS two times. | ||
4. Add | 4. Add the appropriate pfu/cell (MOI) of the virus in 50 ul of PBS on top of the cells. | ||
5. Incubate at 37°C for 30 minutes. | 5. Incubate at 37°C for 30 minutes. | ||
6. Add | 6. Add 50 ul of 2x media on top of the cells (don't remove the virus) - you want this media to contain 2x FBS. | ||
7. Incubate for as long as you need at 37°C/5% CO2 | 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. | 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 | PFU = MOI * #cells |
Revision as of 08:22, 14 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 2000 cells per well
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 50 ul of PBS on top of the cells.
5. Incubate at 37°C for 30 minutes.
6. Add 50 ul of 2x media on top of the cells (don't remove the virus) - you want this media to contain 2x 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