I am using RetroNectin to coat plates for viral infection. The protocol (see the link below) says to block with 2% BSA for 30 min and wash with PBS after removing the RetroNectin coating. Does anyone know why these steps are needed, and would it affect the viral infection efficiency if not block/wash?
Also, the protocol says to spin down the virus first, and then add cells. Would it make a big difference if I spin the virus and the cells together? I am afraid that I would lose some virus if spin down the virus first and remove the supernatant.
Thanks for your help!
RetroNectin protocol link:
http://www.clontech.com/US/Products/Viral_Transduction/Hematopoietic_Cell_Transduction/ibcGetAttachment.jsp?cItemId=42487&fileId=6939380&sitex=10020:22372:US
Typically, the BSA is added to reduce non-specific protein-protein interactions, the result is a decrease in noise levels and an increase in specificity, sensitivity, or in this case transduction efficacy. I would recommend to do it, and I have never tried to skip this step.
You have two "classical" way to perform your transduction : the first one is to add the virus spin the plate, remove the supernatant and then add the cells, and second is to first add the cells and then put the virus and spin your plate.
It will depend on what type on virus you're working with and what type of cells also. In my case I used primary CD4 T cells from mice. As I had to make them proliferate first to transduce them, here was my protocol :
Day 0: P24 Coating.
- Coat p24 wells with 3ug/ml of anti-CD3 at least 3H at 37°C.
-Wash with PBS
- Add 40ug/ml (200ul) of Retronectin and incubate O/N at 4°C
Day 1: Stimulation of CD4 T Cells.
- Add 1ml of PBS 2% BSA and incubate 30 mins at room temperature.
- Harvest CD4 T cells.
- Plate cells at 500 000 cells in 1ml of OptiMEM medium containing 2ug/ml of anti-CD28 and 1 UI/ml of IL-2 (It also works with complete RPMI).
Day 2: Transduction.
- 18H after stimulation (Doesn't work well after 24H) replace media by virus-containing media (My virus was in OptiMEM) and centrifuge plates at 3000 RPM 32°C for 1,5 hour (The less volume you put the more you'll increase the transduction efficiency).
- After transduction add 500uL of OptiMEM containing 2X cytokines and CD28 mAbs.
Day 3: Same as Day 2 (Replace medium and put a second wave of virus, spin and add medium containing 2X cytokines
Day 4 : Put cells in complete RPMI medium
- Put cells in Complete RPMI media (+IL-2 + aCD28).
Depending also on what is your readout, you can add anti-IFNg it improves a lot the transduction efficiency in my case but I don't know whether it is because my cells survived/proliferate more or whether it was due to a better transduction efficiency.
I don't know if it helps.
Hi Michael, thanks a lot for your answer. You mentioned BSA is used to prevent non-specific protein-protein interaction and to increase transduction efficiency in this case. But the retronectin is diluted in PBS, what kind of non-specific protein-protein interaction do you think might happen? Or since the virus solution contains DMEM (supposedly diluted in PBS, but some DMEM remains), did you mean that the retronectin will interact with some proteins in DMEM? And thanks again for the transduction protocol.
You'll probably have non specific interactions either with lentiviral particles or with T cells that will interact with other proteins on the cell surface (but not with VLA-5 and VLA-4) making weak interaction with retronectin I suppose
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