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

Hi Everyone!!


I got 15 emails that help me a lot about three color flow cytometry...I want to say that
it was great for my understanding about flow cytometry methods and help me to decide
which color to use...

First of all...It was my bad...I didn't say anything about the machines are available to
me here at CoreLab at University of Florida....I can use a FACSSort and FASCCAlibur and
the last one I will used because it has two laser...I'm sorry...

I've decided to use AlexaFluor 467 that was Neal advice, but all tips were good, if it
doesn't work I'll be able to try other possibilities....

I'm so glad to join this list, because as I said I'm just learning....I have experience
in Flow Cytometry but using as a tool for hybridome selection and two colors assays, when
I did my master in Brazil...

Thanks again...

I'll update you about the results that I hope I will get...

Sincerely

Lilian Oliveira


________________________________

From: alexis@igc.gulbenkian.pt [mailto:alexis@igc.gulbenkian.pt]
Sent: Wed 4/19/2006 7:39 AM
To: cyto-inbox
Subject: Re: Three color flow cytometry!!! Help needed :)



Hi Lilian,

We currently do a lot of measurements of CD25 and FOXP3 in our institute.
For CD25, we have used Alexa488 and PE with good results. As you are already
using this two fluorochromes in anti-CD4 and FoxP3 conjugates, I think you
could try for CD25 another "bright" fluorochrome as Cy5-PE (also known as
Cychrome). In our lab we rather prefer to use antiCD25-alexa488, antiFoxP3-PE,
antiCD4-Cy5PE combination. I think it is the best for compensations to keep
Cy5PE for CD4 (a highly expressed marker): this will minimize the errors that
tend to be higher
while compensating fluorochromes targeting dim (faint) markers as CD25 or FoxP3.
If you want to go in details into how to make a proper choice of fluorochromes
and learn in depth about compensations you could find deep explanations in the
following references:
1- web site on compensation by Dr. Mario Roeder:
http://drmr.com/compensation/

2-A practical approach to multicolor flow cytometry for
immunophenotyping. Nicole Baumgarth and Mario Roederer
Journal of Immunological Methods 243 (2000) 77âEUR"97


All the best and good luck,

Alexis Pérez González, PhD
Flow Cytometry Facility
Cell Imaging Unit
Instituto Gulbenkian de Ciência,
Oeiras, Portugal


Quoting "Oliveira, Lilian" <oliveira@animal.ufl.edu>:

> 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
>
>