I would like to discover the effect of my target gene on macrophage polarizing. I have used lentivirus to infect PMA-treated THP-1 to konckdown my gene, but it was too difficult to be infected. Can I infect THP-1with the lentivirus before it is differentiated to macrophage? It may be easier to knockdown my gene in suspended THP-1 cells.
Hi Gao Yue,
Do the THP-1 already express the surface markers necessary for your lentiviral vector to infect? Normally, monocytes (to which THP-1 are close) can be infected like macrophages, but I'm not sure about the specifics of your viral vector. Maybe this paper could be of interest:
Article Expression of CD4 Controls the Susceptibility of THP-1 Cells...
I hope this might help,
Kind regards,
Jasper
I can understand this difficulty.
You can try the following to better infect the monocytic THP-1 using Lentivirus.
1. Cote the plate with poly lysine - monocytes then can loosely adhere to the surface and will be better infected.
2. Use high titre lenti preparation for infection, there are methods to generate high titres.
3. you can calibrate the amount of polyberene used to for infection in terms of amount and pre-incubation before infection.
I wish this will help you to overcome.
Good luck!
I used lentivirus to infect THP-1 cells, after FACS, then add PMA to make it differentiated to macrophage .
Yes, you can transduce THP-1 before PMA treatment, they are quite easy to transduce. After adding lentivirus and polybrene (in the final concentration of 8ug/ml) you need to spinfect the cells (800g, 45 min, 37 C).
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