I have a lentivirus (VSV-G) containing an enhancer and general promoter coupled to a fluorescent protein-encoding gene (mCherry). The vector is used as a tool to identify factors able to stimulate the enhancer. In unstimulated cells fluorescent protein should not be expressed.
I followed this protocol:
Day -1: Trypsinize cells and plate in 24-well plate with lentivirus-containing medium (MOI 5).
Day 0: Stimulate cells
Day 3: Determine positive cell fraction by flow cytometry.
% postive cells was 0, although I know that a certain combination did work before (using a different lentivirus containing GFP but with the same enhancer).
I thought it could be possible that the infection is not occuring because the cells miss the receptor (due to trypsinization) necessary for viral delivery.
Hello Rajiv,
I am not sure if I understand your question properly. I think you are asking if you can infect the cells before plating but after trypsinizing? How will that bypass the effect of trypsin you hypothesized?
Alternatively, you could try to use milder cell disassociation enzymes like TrypLE.
Hi Sajjeev,
Thank you for replying. Basically the experiment failed and I'm troubleshooting to find a explanation for it. One possibility is that the virus infection didnt occur and one explanation for it could be that trypsinized cells are missing the receptor that the virus needs to enter the cell.
In my previous experiments, I first plated the cells and did the viral infection a day later when the cells are attached again.
You are welcome, makes sense now. Waiting for the cells to attach would be the right thing to do as it would give the cells sometime to recover from the stress. I would try three things
1) Use Trypsin alternatives
2) Titer the virus and try to optimize using various amounts of lent viral particles.
3) Making sure your stimulant is working properly.
Good luck
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