Transduction of THP-1s

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

1. Count cells, add cells to appropriate size tube, spin down at 400 x G for 5 minutes
2. Re-suspend cells in the transduction reaction mixture
3. Spin at 400 X G, 10 degrees for 1 hour 30 minutes
4. Re-suspend the cells in the virus containing media and transfer to an appropriate size flask or plate, incubate overnight at 37 degrees overnight
5. Transfer the contents of the flask to a new tube, spin down and replace the media with 500 uL of fresh complete RPMI
6. 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