[Cytometry] Indo-1 on an Aria?

Shaw, Pam pam.shaw at mso.umt.edu
Fri Feb 20 15:45:15 EST 2009


Hi Ray,

We don't have a UV laser on our Aria, so we can't use the Indo dye, but
we have been able to use other dye(s) to do Calcium Flux.  There is a
protocol in "Methods in Cell Biology" by Carl June and Peter
Rabinovitch.  You can also find the protocol in "Current Protocols in
Cyotmetry."  It covers the Indo dye as well as other combination dyes
(Fura Red and Fluo-3.)  We are currently working on developing a
protocol that uses Fura Red alone based on it's unique properties of
being excited at two different wavelengths depending on its calcium
state (bound or free.)  

The nice thing about the Aria is that it has the option of warming the
bulk injection chamber which is required in the protocol.  The problems
we have run into is when you unload the sample, add your compound and
reload, it takes a while to stabilize the signal.  If your flux is
immediate and not long-live, you can miss it.

If you can't find the protocols, I can find you some references.  Try
doing a search for Peter Rabinovitch - he has several articles on the
method.

Pam Shaw
Fluorescence Cytometry Core Facilitator
Center for Environmental Health Sciences
The University of Montana
32 Campus Dr, Skaggs Building 052
Missoula, MT 59812
 
Ph (406) 243-4974
Email:  pamela.shaw at umontana.edu
www.umt.edu/cehs/fluor_main.html 
-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Ray Hester
Sent: Thursday, February 19, 2009 8:59 AM
To: Cytometry Mailing List
Subject: [Cytometry] Indo-1 on an Aria?

Hi,

Has anyone demonstrated calcium flux on a FACS Aria or Aria II (_not_ a 
FACSVantage) where at some point after equilibration of Indo1-loaded 
cells, the sample tube was removed from the sample uptake tube, an 
agonist (or some compund) added to the tube, the sample tube replaced 
and the experiment continued?

If so, or if you know where this type of experiment on an Aria has been 
published, can you point us to references (journal articles) detailing 
the experiment/s?

Thanks.

Ray Hester
Research Cytometry Laboratory
College of Medicine
The University of South Alabama
Mobile, AL 36688
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