[Cytometry] Signal preservation of fluorescently stained cells on FACS/microscopy

Subhash Kulkarni skulkarn at stanford.edu
Sun May 8 16:12:32 EDT 2011


As is evident from the subject, I am trying to find ways and means of
preserving the signal intensity of cells stained with fluorescently
labeled antibodies so that I can use FACS or do live cell imaging on a
microscope, without a significant loss in signal.
Does anyone have any experience with this?

Please do let me know

Regards,
Subhash

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Mario Roederer
Sent: Monday, May 02, 2011 4:20 AM
To: Jasneet Kaur Phd2010
Cc: cytometry at lists.purdue.edu
Subject: Re: [Cytometry] PE query

I wouldn't use PE.  Given essentially no details about what you are trying
to accomplish, it's virtually impossible to suggest much. But I can't
think of an experiment where I would use PE.

If you are studying non-specific endocytosis (pinocytosis), which is about
the only way PE would be taken up, you would need to use high
concentrations to get signal.  But then the problem is that the
proteolytic environment of the early endosome (and lower pH) will start
degrading the PE and you will lose signal.  And to use PE for this is
phenomenally expensive.  Instead, consider using a conjugated dextran (eg
from Sigma, I think), which you can use economically at the nearly mg/ml
levels you need for pinocytosis experiments.

As for "all cell types", the answer there is also no.  Every cell type is
different.

If you are just trying to stain cells without relying on an active
procees, consider using a membrane probe like Nile Red.  Again, very
inexpensive and it WILL label all cells.

mr


On May 2, 2011, at 12:48 AM, Jasneet Kaur Phd2010 wrote:

> hi all,
> 
> i have a Phycoerythrin dye in my lab (not conjugated to anything)
obtained
> from Molecular Probes. i have to use a dye to just stain a cell
population
> with it. does this dye enter/bind to all cell types without any
preference?
> also, if possible, can anyone send me the protocol for its use? i am
> completely clueless about the concentration/duration of the treatment.
> thanks in advance.
> 
> -- 
> Jasneet Kaur
> c/o Dr. Satyajit Rath
> Research Scholar
> Immunobiology Lab-2
> National Institute of Immunology,
> New Delhi
> _______________________________________________
> Cytometry mailing list
> Cytometry at lists.purdue.edu
> https://lists.purdue.edu/mailman/listinfo/cytometry
> Search the list archive at  http://tinyurl.com/cytometry


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