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Quantifying bacterial infection microscopically


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#1 virusfan

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Posted 06 August 2010 - 12:02 AM

Hi all,
I'm not sure if this is the right column to post my question.

I transfected my cells with GFP-tagged cellular markers, which usually ended up with low transfection efficiency (~15%). Then, I infected these transfected cells with my fluorescent bacteria, imaged them live with a confocal microscope. Several fields of the images were taken.

My objective for my experiment is to examine the effect of the cellular markers (wild type and mutants) on the bacterial invasion of the cells. Therefore, for quantification, I counted total number of GFP expressing cells with intracellular bacteria, divided by the total number of GFP expressing cells (both uninfected and infected), multiplied by 100%. I addressed the percentage from my calculation as the percentage of infection, and used the resulted values to conclude the effect of the mutant cellular markers on the bacterial invasion.

However, I was told that my calculation is wrong. The reason is I did not correct my calculation with the cells transfection efficiency.

In my own opinion, I don't see anything wrong with my calculation. Therefore, I would like to seek your opinion on this.

Thank you.

Cheers.

#2 bob1

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Posted 10 August 2010 - 02:44 PM

Your calculation looks OK to me so long as you were only looking at the GFP expressing cells.

Just as an aside... How do you know that the GFP tag isn't interfering with the protein in terms of folding or steric hindrance etc.?

#3 virusfan

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Posted 17 August 2010 - 09:36 AM

Your calculation looks OK to me so long as you were only looking at the GFP expressing cells.

Just as an aside... How do you know that the GFP tag isn't interfering with the protein in terms of folding or steric hindrance etc.?


That's tricky. However, those constructs have been used by others and they have shown that the fusion protein is functional.
Another control I included was the empty gfp plasmid, which express the gfp only.

Cheers. :)




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