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Calculating MOI for luciferase-transducted cells.


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#1 maxrempel

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Posted 27 April 2009 - 12:18 PM

This is likely a very simple question. I just don't know how to Google it, so please give me a brief intro...
I need to measure MOI (multiplicity of infection) for a viral vector carrying a luciferase gene.
I will be infecting adherent cultures in 24 well plates.
I know that for GFP, I would just look at GFP fluorescence in a fluorescence microscope.
But how would I see the Luciferase-expressing cells?
I assume, I need to add a substrate, which will be taken by cells and they would luminescence.
Would the sensitivity of a fluorescent microscope be enough to see the signal?

#2 bob1

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Posted 27 April 2009 - 05:38 PM

GFP will glow under normal light.

Anyway, if you are infecting with a pure stock of the virus (i.e. not in conjunction with another virus), then just assess the MOI as you would normally. The GFP doesn't do anything special.

#3 dpo

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Posted 28 April 2009 - 04:50 AM

I've only done luciferase measurements on lysed cells, but I assume you can incubate cells with luciferin and detect luciferase activity. However, you can't use a fluorescence microscope for this, as the luciferase produces light on its own. With mice for instance, you can inject the substrate and then detect luciferase activity with a sensitive camera, but I don't know if the signal of your cells will be strong enough to see without such a camers ...

#4 maxrempel

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Posted 29 April 2009 - 09:42 AM

..

Edited by maxrempel, 29 April 2009 - 09:42 AM.





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