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lentivirus tranduction of monocytes


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#1 hoshi

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Posted 07 July 2009 - 09:27 AM

Hey all,
I understand monocytes are difficult to infect with lentivirus constructs but I am looking for a way to improve efficiency of our lentivirus transductions of human monocytes.

Monocytes are isolated by plastic adherence from PBMCs in 12-well plates. Then cells are cultured in about 300ul RPMI +10% FBS +2-5ug/ml polybrene/well before adding LV virus overnight and adding more media the next morning. We have tried just adding the virus directly to the media as well as spinoculation techniques with no success. I've also done a general google/Pubmed search for methods but have not found many suggestions for overcoming the problem.

Is there anyone who has any experience in this area or can point out a reference please?

#2 cellcounter

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Posted 07 July 2009 - 09:34 AM

Hey all,
I understand monocytes are difficult to infect with lentivirus constructs but I am looking for a way to improve efficiency of our lentivirus transductions of human monocytes.

Monocytes are isolated by plastic adherence from PBMCs in 12-well plates. Then cells are cultured in about 300ul RPMI +10% FBS +2-5ug/ml polybrene/well before adding LV virus overnight and adding more media the next morning. We have tried just adding the virus directly to the media as well as spinoculation techniques with no success. I've also done a general google/Pubmed search for methods but have not found many suggestions for overcoming the problem.

Is there anyone who has any experience in this area or can point out a reference please?

Monocytes are difficult to transfect/transduce. You may want to try out retronectin coating of the plates, though I am not sure if it works.

#3 hoshi

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Posted 07 July 2009 - 10:55 AM

I've tried retronectin for CD34+ HPCs but it didnt help LV infection efficiency in my hands. I can see if we still have some around the lab. thanks.




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