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

Ruud Hulspas rhulspas at cytonomest.com
Sun May 8 17:10:44 EDT 2011


Subhash, I assume you mean "..FACS AND live cell imaging on a 
microscope.." (not OR).
Which of the two technologies do you need to do first ? If you have a 
choice, I would do microscopy AFTER flow cytometry. I have never had any 
problems seeing fluorescent, labeled areas on sorted cells. There maybe 
some loss in signal intensity due to exposure to laser light, but this 
is compensated by the fact that the eye is much better capable of 
distinguishing background from signal than a flow cytometer is.

Ruud

On 5/8/2011 4:12 PM, Subhash Kulkarni wrote:
> 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
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>
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-- 

*Dr. Ruud Hulspas, Ph.D.*
Scientific Director

Cytonome/ST, LLC
Cytonome/ST, LLC
27 Drydock Avenue
Boston,  MA 02210
phone: 617-330-5030 x226
fax: 617-330-5031
RHulspas at cytonomest.com <mailto:RHulspas at cytonomest.com>

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