HEPES and retroviral transduction - (Sep/28/2006 )
Hello,
Has anybody observed that the addition of HEPES can adversely affect retroviral transduction efficiency.
I am generating viral supernatants through the transfection of 293T cells with my construct and helper construct. I have been trying to test the supes on 3T3 cells, and have gotten very inefficient transduction. This used to work for me. The main thing that I think that I have switched is that recently, I have started to add HEPES to my DMEM (as without it, the media was turning pink, rather quickly). Could that affect transduction? I do add polybrene (4ug/ml) to the mixture; my thought was that perhaps the buffering activity of the HEPES affects the activity of the polybrene. I appreciate any input
Michael
I didnt understand what exactly u mean by adding HEPES to DMEM.
How r u transfecting ur 293T cells ? And which point in the transfection step r u adding HEPES?
i have tried polybrene and wasnt of much use for my cells. later, I learnt that polybrene might not aid all cells only certain cell types. and one has to optimise the right conc.
Bytransduction, do u mean infection of cells with viral supernatant?
How r u transfecting ur 293T cells ? And which point in the transfection step r u adding HEPES?
i have tried polybrene and wasnt of much use for my cells. later, I learnt that polybrene might not aid all cells only certain cell types. and one has to optimise the right conc.
Bytransduction, do u mean infection of cells with viral supernatant?
To transfect my 293T cells, I have been using Fugene 6. That has worked fine for me. I then harvested the supernatant, which I used to transduce 3T3 cells, in the presence of polybrene to determine the transduction efficiency. In the past, this also has worked fine. Recently, I have had some problems, and am trying to think of things that I have altered.
Regarding the HEPES addition, I meant that in the past I had been using DMEM, with the addition of FBS, L-glutamine, and Penn-Strep to grow the 3T3cells. I found that the media that I had been storing that way changed color (ie pH) rather quickly. I was advised to add 10mM HEPES as a buffering agent. I have been doing that, and my cells are growing fine. But I am wondering that if the addition of HEPES to the media could affect the retroviral transduction.
I am not sure if HEPES could interfere with polybrene. But as HEPES changes the pH, this could affect transduction itself by interfering with binding of viral particles to the cell membrane. And since polybrene, a charged molecule, was aiding transduction, small changes in pH could alter its funcitoning as well.
i think there is a paper which suggests that slight increases in pH could help transduction efficiency.
I frequently resuspend retrovirus in HEPES buffer, so this should not matter unless the pH is way off. Check to make sure you are producing a virus with the correct receptor (amphotropic vs. ecotropic).