ara wrote:
> Hi, there!
>> I have a question : Do the HeLa cells which are not transfected with GFP
> gene (From
> now on, I will call these 'control HeLa') have auto-fluorescence? (FL1
> emmision)
> If they do, what's the intesity value of them? (I mean 'mean value')
>> I transfected HeLa with pEGFP-C1 containing GFP gene (35 times more
> intensely than WT). and I had the cytometrical data using FACS after 48
> hrs.
> When compared with control HeLa, transfected HeLa has not so high
> intensity.
> And control HeLa has already somewhat high intensity value.
> However after treatment of 70% EtOH, control HeLa as well as transfected
> HeLa
> has low intensity value.
> Is there anybody who can explain why?
>> Thanks in advance,
>> Sincerely yours,
Hi!I would like to make several comments based on my experience with FACS:
1. All cells have some autofluorescence. Bigger cells often have more of it.
We often observed higher fluorescence in "unhappy" cells (drug treated,
etc.).
2. The absolute fluorescence value you get from FACS depends on the settings
on your machine, and with nothing to compare to it makes no particular
sense, e.g. you cannot call it "high" or "low". You can lower it, for
example, by lowering amplification or voltage in your detectors.
3. Autofluorescence may also depend on conditions of cell culture and the
diluent used to resuspend cells before analysis. Some people suggested to
lower the amount of potentially fluorescent compounds in growth medium. We
normally run samples in PBS, as it gives us less fluorescence in naive
cells.
4. Autofluorescence of your sample maybe somewhat decreased by selecting
optics that would allow efficient detection of your molecule of interest
(e.g. GFP), but decrease the chances of detecting the "contaminating"
fluorophores.
5. As detection of positive cells by flow cytometry is usually done
relatively to the negative control, lowering autofluorescence of negative
sample can, indeed, make positive cells look brighter.
6. After EtOH treatment some content of a cell (including GFP) may leak out.
Also, cells shape and size is likely to change. Thus, the change in
fluorescence that you observed is not surprising at all.
7. EGFP is usually so bright that we have to make an extra effort to fit
positive and negative cells on the same scale. If you have problem detecting
it, I would wonder whether your transfection worked.
Eugene Kandel
U09577 at uic.edu
