RE: Three color flow cytometry!!! Help needed :)

Hi Lilian,

Nice to hear someone's validated Zenon for intracellular; I've never
tried that myself yet.

Multicolour with two Zenon reagents is usually not a problem if you
don't let the stained samples sit around too long before analysis (even
if the two primaries are the same species/isotype, but try to avoid it
if you can just so this won't raise doubts about "double positives"
afterwards).

If would be very useful if you told us which instrument you'll be
measuring with; most will take FITC/A488 and PE combo's, but your
optimal third colour choice may depend on the instrument (red laser
present yes/no? do you include live/dead discrimination and if so, what
dye?). Even assuming you're working with a standard benchtop analyzer,
which few people modify, just make sure no standard filtersets have been
replaced with others in your instrument, or any recommendations will be
off as well.

Apart from theoretical considerations re: non-covalent conjugation of
antibodies, have you considered conjugating your antibodies yourself?
Zenon's pretty expensive, and conjugations are not that hard given some
time and basic protein purification/QC resources.

Best of luck,

Guy

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Guy Hermans, PhD
Senior Scientist
guy.hermans@ablynx.com
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-----Original Message-----
From: Oliveira, Lilian [mailto:oliveira@animal.ufl.edu] 
Sent: Tuesday, April 18, 2006 3:29 PM
To: cyto-inbox
Subject: Three color flow cytometry!!! Help needed :)


Hello everyone!
I'm Lilian Oliveira and I'm trying to study Treg cells in livestock
animals, so I have a very basic question for you about which
fluorochrome I should used in my experiments. I have a anti CD25 that is
IgG2a unconjugated, and I want to label my cells for FoxP3 and CD4 just
to start to search these cells in animal model. My FoxP3 antibody is
labeled with Zenon Alexa fluor 488, and my CD4 is PE labeled....I really
don't know which Zenon labeling I have to buy in order to proceed three
color studies....if somebody can help me... I'm a Ph. D. student, and
I'm starting to work with Flow Cytometry....I'm so glad to enjoy in this
list, I'm learning a lot with you.... Thanks in advance Lilian Oliveira