dkc

Hello,

I have been attempting to produce lentivirus in 293FT cells, I have been using normal DMEM + 10%FBS as my regular culture, transfection, and viral production medium.

I've noticed that after I transfect the 293FT's and replace the transfection media with fresh media for viral production, the media becomes acidic much more rapidly than usual.  I have read that this acidic pH can be harmful for viral infectivity, do others have this problem with the viral production media becoming acidic too rapidly?  Have you noticed this affecting your titers or infectivity?

I recently tried supplementing the culture medium by adding HEPES to a concentration of 25 mM in order to buffer against the acidity, however I noticed that after adding the HEPES to the DMEM it becomes more orange-ish than it is normally, meaning it is starting at a more acidic pH than the DMEM without HEPES.  Do I need to re-titrate the media after adding HEPES? I was under the impression that it would inherently balance itself based off its pKa, however this doesn't seem to be happening.

Thanks for your time!
-dkc

Hello,

A lot of the commercial vendors for lentivectors have their own proprietary packaging mix that their manuals claim you must use in order to get packaging of the protein into working viral particles (e.g. Clontech's pLVX and lenti-X HTX, or Invitrogen and virapower, or SBI and pPACK, etc.)

I currently have both 2nd and 3rd generation packaging systems but none of them are the proprietary fancy name kind, my question is if I can use these with a commercial lentivector and still get good packaging into viral particles?  Or, are the commercial lentivectors altered in some way that they MUST be packaged with the proprietary packaging mix from the same company.

Thanks,
-dkc