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What does the shift in G0/G1 tell us?


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6 replies to this topic

#1 Curtis

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Posted 02 January 2011 - 06:13 AM

Hi,

I transfected my normal cells with killer plasmids and after cell death I ran a flowcyto and histogram analysis.

my boss asked me 'what does the shift in G0/G1 tell us?'. I had read somewhere that if the number of readings for each sample is different then the peaks shift, but in here the number of all readings is constant (1000 events).

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#2 Rsm

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Posted 04 January 2011 - 02:35 AM

That depends on what your labelling with APC-Cy7 is... some more details? Cell cycle? Apoptosis? Surface molecules?

rsm
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#3 Curtis

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Posted 04 January 2011 - 03:46 AM

I just transfect HeLa cells, and after 18 h I stain with PI and read by flowcyto machine, just very simple.

the blue curve is untransfected cells, and the red is complete dead cells as control. The green and yellow are my transfected samples.

#4 Rsm

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Posted 04 January 2011 - 08:00 AM

I just transfect HeLa cells, and after 18 h I stain with PI and read by flowcyto machine, just very simple.

the blue curve is untransfected cells, and the red is complete dead cells as control. The green and yellow are my transfected samples.


Do you fix your cells before analysis? I am still a bit confused what you detect in the APC-Cy7 channel. It can't be PI, because then live cells should be negative, and dead ones positive, not the other way round. Maybe someone else has an idea?

rsm
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#5 Curtis

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Posted 04 January 2011 - 08:54 AM

yes, I fix them with Ice cold Ethanol. Don't mind the channel, it was the same for the other channels too. The G0/G1 shifts in alexa 488, PE or others.

#6 DRT

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Posted 05 January 2011 - 02:58 PM

Is the number of cells being stained roughly similar? If the PI is sub-saturating then the sample with fewer cells will positive shift because of a greater PI/cell.

#7 Denis Baev

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Posted 19 January 2011 - 03:22 AM

If you're using PI for cell cycle together with other fluorochromes in multicolor you coud have some comensation problems beacuse of PI-s wide emission spectra. The best way to avoid that and to make your "flow life" easier is to replace PI by DyeCycle dye or by 7-AAD




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