Flow cytometry

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Flow cytometry

Postby researchwoman » May 27 2010 5:56 pm

Hey all
I have a question about flow cytometry. As shown in the attached figure, my 'untransfected' or control cells where I measure the basal amounts of fluorescence o normaize, appears very close to the y-axis. Is this acceptable or was I to have adjusting instruments settings in order to get the 'whole peak' in the visible scale?
So the values given in the percentage total or gated- s it reliable or represents only a random(?) fraction of cells that were picked up in that area in the untransfected versus siGlo treated cells?
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Re: Flow cytometry

Postby CrowSan » Jun 04 2010 3:04 pm

Hi if I understand you correctly you are worried that the "shoulder" in the negative control in the M1 gate is less than the "shoulder" of the negative cells in your treated cell population? Personally I would increase the PMT on the forward scatter to pull the untreated cells forward a bit (or drop the PMT on the siG. treated cells) to make the two graphs more equivalent.
However it depends what you are trying to say with them. ...
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Re: Flow cytometry

Postby researchwoman » Jun 10 2010 1:08 pm

I wanted to see the whole peak...what is PMC?
I acquired again to get the peaks inside the graph.

Thank you...
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Re: Flow cytometry

Postby CrowSan » Jun 10 2010 2:17 pm

"PMT" - sorry it means "photo-multiplier".
I think the forward scatter (x axis) is on PMT1 and the side scatter (y) is on PMT2 (although it has been greater than 6 years since I've used a facs so i could be wrong). To shift the peaks to the right just increase the PMT whilst the cells are running through the machine until the desired position is reached then re-start the count - but it sounds like you've solved the problem anyway :)
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Re: Flow cytometry

Postby researchwoman » Jun 14 2010 1:57 pm

Yes. Indeed thats what I did. Didn't know the name. We call it 'gain'.
Thanks.
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Re: Flow cytometry

Postby Gnapponico » Jun 14 2010 9:04 pm

Pay attention on PMT and gain...they are not the same thing...
and...FSC is usually a photodiode, not a photomultiplier!
If you are in a log scale (as usual for FL1, so, fluorescence) you can only move PMT, not the gain.
You can move the gain and the PMT only on linear scales (i.e. SSC only).
For FSC you can move only the gain (because of photodiode "structure").
To stay on topic, for your next acquisitions... first you have to move your population: move up the SSC voltage (PMT up); FSC is quite good, but you have to use the linear scale (in the image I see X and Y axes as logarithm).
Move the FL1 curve till you have a nice histogram between 10^0 and 10^1 (more or less) for the negative control, then acquire your samples...
hope it can help...

Ps:
So the values given in the percentage total or gated- s it reliable or represents only a random(?)

In your image (and in the analysis too) you don't have any gate...the value is reliable
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Re: Flow cytometry

Postby researchwoman » Jun 15 2010 10:13 am

Thanks...I got the 'control' peak fully within the graph now...
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