Summary Document

This document is a summary of message from the Purdue University Cytometry Laboratories Cytometry Discussion list.

http://www.cyto.purdue.edu/hmarchiv/index.htm

Thanks to all of you that answered my question on fluorescent probes, I
have listed the replies below in case any of you didn't get them all.

Thanks again

Dr Rachael Walker
Flow Cytometry Core Facility Manager
Wellcome Trust Centre for Stem Cell Research
University of Cambridge
Tennis Court Road
Cambridge
CB2 1QR

01223 760227

Responses:

I do a similar analysis using cells on glass which involves automated cell
recognition and measurement of fluorescent intensity with a software package. For
individual cells I have found that a long exposure time (2 to 5 seconds) on the
microscope can be enough to visualize cell membranes by auto fluorescence and allows the
software to automatically recognize a cell. Perhaps this may be useful for your
application and you can avoid probes all together...

Mara Rocchi wrote (to me, not the Mailing List):
> Do you have a reference protocol or paper I can look at to use the dyes
> you suggested for live staining?
See below.
We need to stain effectors or targets cells in a CTL assay, and I had
> toxicity problems using CFSE in the past.

I had posted the following in response to Rachael Walker:
Your best bet might be a reactive dye such as FITC, Cy3,
> or Cy5; these covalently label structure on the cell exterior but do not
> get inside intact cells to an appreciable extent. You can pretty much
> pick a dye with spectral characteristics that won't interfere with
> stains you might want to use later, and the reactive dyes are typically
> nontoxic at concentrations that will give you the staining you need.
>

I suspected that the work I vaguely remembered was decades old; it is. I
am posting the references (from Practical Flow Cytometry) in case others
might be interested. Newer relevant references would be appreciated.

1540. Abernethy NJ, Chin W, Lyons H, Hay JB: A dual laser analysis of
the migration of XRITC-labeled, FITC-labeled, and double-labeled
lymphocytes in sheep. Cytometry 6:407-13, 1985

1541. Capo C, Mege JL, Benoliel AM, Mishal Z, Bongrand P: Quantification
of the nonspecific intercellular transfer of fluorescent molecules
between labeled and unlabeled rat thymocytes. Cytometry 8:468-73, 1987

Since these are both from Cytometry, they are accessible online free to
ISAC members.
-Howard

As Howard says, reactive dyes are a good way to go, but I happen to have a
bucket of fluorescein diacetate which will pretty much only label the viable
cells, which may be of use if the colonies you're picking need to be in good
shape (it's cleft by the mysterious and wonderful non-specific esterase
activity in your average cell, and retained as a charged thing by most
intact cell membranes - ES cells may extrude free fluorescein, I'm not sure,
but that may be a bonus too if coupled with a surface-labeling probe such as
those Howard suggests ), - it doesn't do much lasting harm as the free
fluorescein, and will even leak out eventually, give me a bell or pop over
and I'll give you a gram or two to play with,

but....be somewhat careful with AO - it's a great stain, but if you are
not careful, you will stain the tubing into your cytometer as
well...then you can be sure that everything will be positive for a
while...even when they are not..!
So take care with the concentration and run some control cells before
and after your sample just to check that the dye is not persisting in
your machine.

pkh26 will stain cell plasma membranes orange and CFSE will stain
cytoplasm green, as will FDA. Toxicity is a tricky question but we have
used pkh26 and FDA (separately) with no obvious toxicity or change in
cell behaviour. Both will require incubation but this should be
"doable".

Hi - I wonder if the dyes CFSE or CFDA, which are membrane bound & only fluoresce when
metabolized would work?
Cells in suspension have to be incubated w/ the dyes for I think at least 20 mins, so
maybe that is not instantly enough for your needs.

acridine orange. stains cells bright green, just mix dye with cells and you are good to
go

It would be rather surprising if the Vybrant dues were to interfere with DNA,
because they are highly lipophilic and ought to partition almost entirely just
in to lipid (for most cell types this means membrane bilayers). I would try
one of DiI, DiO or DiD depending on the spectral characteristics you want
(offered by Mol Probes http://probes.invitrogen.com/media/pis/mp22885.pdf ).
It's the organic solvent they come in (EtOH or DMF) that I'd be worried about.