Does anyone have experience in infecting suspension cells (like THP-1 or K562 etc) by retrovirus?

Dear Hanif!

Please You look at this protocol:

https://systembio.com/wp-content/uploads/Spinoculation_protocol-1.pdf

https://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/shrna/learning-center/spinoculation-protocol.html

Protocol

Determine the final puromycin concentration to be used in the selection. The exact concentration is cell line dependent and should be determined by a 72 hour cytotoxicity assay on non-transduced cells.

Utilize only sterile items and work using standard tissue culture sterility techniques throughout this procedure.

Pre-warm all media to 37 °C.

Count cultured viable Jurkat cells.

Dilute Jurkat cells into complete medium to a final concentration of 1x105 cells/mL with a final volume of 14 mL.

Transfer 2 mL of 1x105 cells/mL into each of six sterile 15 mL conical tubes.

Add MISSION® lentiviral particle solution to the Jurkat cells such that the final multiplicity of infection (MOI) equals 0, 0.1, 0.5, 1.0, 5.0, or 10 per conical. The 0 MOI represents your non-transduced negative control.

Centrifuge the cells at 800 x g for 30 minutes at 32 °C.

Virus containing medium is aspirated and disposed of appropriately as per your institution.

Each cell pellet is resuspended in 2 mL of media by gently pipetting the pellet up and down, and each resuspended pellet is transferred to its own well in a 6-well plate tissue culture plate.

The plate is returned to the tissue culture incubator for three days.

After 3 days, transfer cells to 6 separate 15 ml conical tubes and pellet at 200 x g for 5 min. Remove media and replace with 2 ml complete media containing puromycin.

Return tissue culture plate to incubator overnight.

Cells are maintained in puromycin until the non-transduced negative control cells have died as determined by inspection or Trypan Blue staining.

Notes

Use the equation below to calculate the MOI in a well. The ViralTiter will be provided in your certificate of analysis. The VolumeofVirus is the volume in mL that is added to the well/centrifuge tube containing you the cells to be transduced. The NumberofCells is equal to the total number of viable cells in the well/centrifuge tube.

References

O'Doherty, U.; Swiggard, W. J.; Malim, M. H. J Virol 2000, 74, 10074-10080.

Andrew Lewis

Retroviruses can be used for transduction of suspension cells. You can pre-coat a non-tissue culture plate with a retronectin solution (for example Takara - see their product and protocol). This can then bind the retrovirus (usually produced in 293T) to the plate. Then centrifuging a plate at around 300-500 x g for 1 hr with your cells, and additional culture time without shaking the plate in an incubator, will bring the cells into contact with the virus to enhance transduction. I have transduced similar cell lines to THP-1 and K562 with 80+% transduction efficiency. Just make sure to produce a retrovirus with the correct tropism (for example amphotropic) so that it will infect human cells. Lentivirus can be advantageous to transduce low-proliferation cells, but with cell lines like THP-1 and K562, as long as you are keeping them healthy in a culture, they should be transduced by retrovirus with little to no issue. Good luck Hanif

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