-----Original Message-----
From: Syed, Farhan A. [SMTP:Syed.Farhan@mayo.edu]
Sent: Thursday, August 08, 2002 9:34 AM
To: Cytometry Mailing List
Subject: RE: darned autofluorescence
Dear Joanna ,
Although I have'nt tried it yet,from what I have read ethidium mono azide can be
used to stain dead cells in intracellular flow, I feel it is possible and acceptable
to stain some cells from the whole prep with ethidium monoazide and locate their
position in a forward Vs side scatter and get them out in analysis on other cells
(other treatments, stains ) from the same sample.If you are using FITC /PE I think
using ethidium- mono azide asa third color will not be practicable due to spectral
overlap. I hope this method sounds logical.Please comment!!. I have some questions
too with regard to intracellular flow which I will shoot off
> Dear Flowers,
> My greetings to all of you, I am new to the group and let me introduce myself, I am
> a Post Doctoral fellow in Endocrinology at the Mayo Clinic and have started to mess
> around with flow cytometry (It also reads the other way around!!!) . I look forward
> to gaining a lot of information and flow- knowhow from you experienced folks. I think
> this list is a great Idea and I have gained considerably already from mails going back
> and forth. well heres my first salvo....
> ...,I am working with human peripheral blood cells I get a high background with the
> isotype ( i am supposedly using the best isotype for an IgG1 Ab - MOPC-21 from sigma)
> I will really appreciate if I could get inputs on blocking reagents to be used .
>
> Thanks
> Farhan Asad Syed,
> Senior Research Fellow,
> Endocrinology Research Unit,
> St. Mary's Hospital,
> Mayo Clinic,
> Rochester,MN
> USA
>
> > -----Original Message-----
> > From: Joanna Roberts [SMTP:jroberts@malaghan.org.nz]
> > Sent: Wednesday, August 07, 2002 12:11 AM
> > To: Cytometry Mailing List
> > Subject: darned autofluorescence
> >
> > Hello flow people,
> >
> > I have a question about autofluorescence that I> '> d appreciate help with.
> > If you see a lot of autofluorescence in samples prepared for intracellular staining
> (fixation, permeabilisation), is dye exclusion of dead cells a way to try and
solve this?
> >
> > { The experiment prep involves enzymatic digestion(with collagenase and DNAse)
> and then percoll gradient of lung samples before intracellular staining.}
> >
> > Any help or advise and tips is much appreciated,
> > Joanna
> >
> > Malaghan Institute
> > Wellington
> > NZ
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