I use lentivirus to deliver shRNA to knockdown my target genes. After 48h post transduction, i use puromycin to select for 24h and then rest cells for another 24h. Then, PMA (100ng/ml) was added to differentiate it into macrophages, but most cells didn't stick to plate and undergo death after 24h, including cells infected with shCtrl lentivirus.
Anybody know how to deal with this problem?
Thanks!
If your transduction worked well (I didn't really understood if it did or not) then your PMA concentration is too low. By the way, how do you prepare it?
Then, if it still doesn't work with higher concentration, the may be you have to avoid resting them for 24h after selection. May be they are functional just after and when you add 24h of resting, they've already consumed all their potential.
Hi Hakim
We are sure the transduction worked well because we get some cells be confirmed by WB after puromycin selection. Then PMA will be added. PMA was bought from Sigma and resolved in DMSO. Stock will be diluted in RPMI1640 just before use.
Usually, concentration of PMA to differentiate THP1 cell range from 5ng/ml to 200ng/ml according to previous publications. In our lab, 5-100ng/ml PMA induce differentiation very well for the cells without any treatments.
Thank you for your suggestions and we will try it.
Ok then,
Try using a higher concentration of PMA : 200 ng/mL but incubate for differentiation during 48h, not less. Use two replicates: one with added PMA diluted stock in RPMI and another in simple PBS with nothing added.
If it still doesn't work, I can't help more but remember: no result could mean result. Maybe that the transfection is silencing some useful genes for cells to differ.
Good luck, keep me tuned please.
Hakim
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Infectious Diseases