[Cytometry] GFP expression after lentivirus infection

Van alphen, Floris P VanalphenF at email.chop.edu
Tue Apr 2 09:14:13 EDT 2013


Hi Eda,

When you look at the cells under the microscope, are they in a
dish/culture flask?
If so, you most likely don't use an oil immersion objective.
This means a lot of the fluorescence signal is lost in space, rather than
being guided to your eye

If your cells are on a glass coverslip and you do use an oil objective,
you will see more GFP+ cells
The oil "captures" the fluorescent signal and channels it to the
objective. This is basically what the gel-coupled cuvette in a flow
cytometer does.
Furthermore, a flow cytometer uses a PMT, which can amplify your signal,
something our eyes unfortunately can not.

You can use an extra well in your experiment which you sacrifice for GFP
analysis by flow.
Alternatively, you can culture your cells and do your experiment in a
plate made of confocal microscopy grade plastic.

Best,
Floris



Floris van Alphen, Bsc
Sr. Research Technician
Children's Hospital Of Philadelphia Research Institute
Stem cell core facility
3501 Civic Center Blvd
CTRB, Lab 5100
Philadelphia, PA  19104
Phone: 267-426-0949
Fax: 215-590-3660
Email: vanalphenf at email.chop.edu







On 4/1/13 10:59 AM, "Schreiber, Kathy L."
<schreiberkl at health.missouri.edu> wrote:

>Please see below for a question from an investigator who is in the
>process of joining the Purdue cytometry list serve. If you have any
>ideas, you can respond directly to Eda at neueda at yahoo.com.
>
>I am performing lentivirus infections to a cell line with GFP reporter
>tag.  The results are confirmed by both microscopically and flow
>cytometricaly. After transfection I can detect signals in both machines
>very well . But after infections, I do not have any signal under the
>microscope but I see quite good positive GFP percentage with flow
>cytometry. I have PI for debris and autoflourescence and negative and
>positive controls for flow .Everything looks right.
>
> I could not figure out the reason. Could you please help me.
>
>I appreciate your help,
>
> Thanks,
>
> Eda
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