Transduction of THP-1s
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Transduction Reaction
- The volume of the reaction is based on the amount of virus available, the number of cells and the MOI being used
- Prepare the mixture as follows:
- X uL of concentrated virus
- X uL of SF Macrophage Media (chose this volume based on the vessel the cells will be grown in
- 1000X polybrene
Protocol
- Count cells, add cells to appropriate size tube, spin down at 400 x G for 5 minutes
- Re-suspend cells in the transduction reaction mixture
- Spin at 400 X G, 10 degrees for 1 hour 30 minutes
- Re-suspend the cells in the virus containing media and transfer to an appropriate size flask or plate, incubate overnight at 37 degrees overnight
- Transfer the contents of the flask to a new tube, spin down and replace the media with 500 uL of fresh complete RPMI
- Incubate plate for 48-72 hours at 37 °C
Notes
- Number of cells: Select number of cells that allows for an MOI of 300, some literature suggests MOI up to 500
- If making a cell line add selection media at the incubation period in step 6. I would also suggest comparing live/dead cell counts for both the control and the transduced cells before every media change to determine if the selection process is working