Does any one have any experience transducing U937, HL60 or K562 cell lines with lentivirus. I am using the plenti-c-myc-ddk-ires-puro vector from Origene (http://www.origene.com/Destination_Vector/PS100069.aspx). Currently I transduce 15000-20000 cells suspended in 0.5 mls by spinoculation (800xg 40 mins 8ug/ml polybrene) on consecutive days with MOIs of 0, 1,2 and 5. Following spinoculation the cells are cultured in 24 well plates in 0.5 mls of media for 3 days. The cells are then transferred to a 96 well plate at approximately 20 000 cells/well in 120ul media supplemented with 0.25-0.5 ug/ml puromycin (conc. determined by 5-7 day cytotox assay). At this point the cells undergo cell death and i lose all the cells within a week.
Does anyone have experience producing stable lentiviral transductions in these cell lines. A protocol and data on the MOIs and transduction effciency might be particularly helpful!
thanks
James
I have previous experience with all of the cell lines you mentioned. Lentiviral transduction of the cells you indicated should be highly efficient, and even without spinoculation should get you high % positive provided you are using enough virus. The protocol you have is fine assuming your virus production is okay. Here are some pointers:
thank you for your reply Qiang Liu
When talking about MOI i am referring to the viral titre as determined by p24 quantification. I have recently suspected I may need to hit MOIs of 50-100 in order to transduce these cells lines and you seem to agree this is the case. In this case i think i will need to concentrate the my viral supernatant?
May anybody have available stable, green or blue fluorescent protein expressing U937 cells
- Badges
- Science method