Dear Laleh,

We currently transduce this type of cell for several experimentations. We transduce the cells in minimal volume: 200ul in 24 wells plate with shaking during 6h. After, we stop shaking and fill with fresh medium to 800ul final. The day after, we change the medium.

We have this type of protocol in our web site. For K562, the protocol is close than the number 1.

http://www.geg-tech.com/Knowledge/protocols/transduction-with-lenti-one

I hope this insight will help you! Let me know if you need more informations !

Best,

Nicolas

Dear Nicolas

thank you very much for valuable comment. I have tried various methods but all failed. I'll try your method. I hope this method will help me. :-(

Laleh,

We work a lot with K562 cells, and do many infections with lentivirus. Could you add more detail about your experiment, especially what lentivector you are currently using? What promoter is used and is there a selection marker? And are you concentrating your virus or not?

In our lab, we concentrate our lentivirus after production using PEG-precipitation. This increases the concentration 50-100x. Then, we "spinoculate" K562 cells with a small amount of virus in an eppendorf tube. Typically, this means putting 50,000 - 100,000 K562 cells in an eppendorf tube, adding 1-2 uL of lentivirus, adding polybrene to 4 ug/ml, and centrifuging for 30 min at 800 x g (at 32 C or room temperature). If your lentivirus is of good quality, this method gives very high infection rate (normally I get greater than 90%...close to 100)

mark

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