(Difference between revisions)
 Revision as of 15:54, 16 July 2013 (view source)← Previous diff Revision as of 10:48, 22 July 2013 (view source)Next diff → Line 1: Line 1: - 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. + 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, 1000 cells/well is good for 48-72 hours. If you're unsure of this number, you can test it over a period of time, or use a couple of seeding concentrations in case one becomes overgrown (so maybe 1000 as well as 500). I also like to incubate my cells in 1% FBS when stimulating to prevent them from overgrowing (cells that grow to confluency after 48 hours will take 72 hours in 1% FBS - but be sure to test this first and make sure your cells can handle it) - 1. Seed approriate number cells per well (for LLC this is 2000) + 1. Seed approriate number cells per well diluted in PBS - 2. Incubate overnight at 37°C/5% CO2. + 2. Incubate for 2 h at 37*C/5% CO2 to make the cells become adherent - 3. Wash cells with 100 ul of PBS two times. + 3. Remove the PBS and add the appropriate pfu/cell (MOI) of the virus in 25 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) - 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) + 4. Incubate at 37°C for 30 minutes. - 5. Incubate at 37°C for 30 minutes. + 5. Add 75 ul of 1.33% FBS media on top of the cells (don't remove the virus) (for 1 ml of 1.33% FBS media, add 13.3 ul FBS + 986.7 ul 0% FBS media) - 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) + 6. Incubate for as long as you need at 37°C/5% CO2 (48-72 hours). - + - 7. Incubate for as long as you need at 37°C/5% CO2 (48-72 hours). +

Revision as of 10:48, 22 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, 1000 cells/well is good for 48-72 hours. If you're unsure of this number, you can test it over a period of time, or use a couple of seeding concentrations in case one becomes overgrown (so maybe 1000 as well as 500). I also like to incubate my cells in 1% FBS when stimulating to prevent them from overgrowing (cells that grow to confluency after 48 hours will take 72 hours in 1% FBS - but be sure to test this first and make sure your cells can handle it)

1. Seed approriate number cells per well diluted in PBS

2. Incubate for 2 h at 37*C/5% CO2 to make the cells become adherent

3. Remove the PBS and add the appropriate pfu/cell (MOI) of the virus in 25 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)

4. Incubate at 37°C for 30 minutes.

5. Add 75 ul of 1.33% FBS media on top of the cells (don't remove the virus) (for 1 ml of 1.33% FBS media, add 13.3 ul FBS + 986.7 ul 0% FBS media)

6. 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