[Cytometry] Indo-1 on an Aria?

Sharma, Parul Parul.Sharma at childrens.harvard.edu
Fri Feb 20 15:00:51 EST 2009


Hi Ray,
We use the calcium sensor BTC AM (Invitrogen, also a cacium sensor)as it works with the 488 laser in my FACSAria. I use Flowjo for the downstream ratiometric analyses.

For BTC ratiometric measurements you should measure the output with the 502LP filter and the 530+/-15 detectors from the 405 and 488 laser.Also set the temperature to 37C on the tube holder.
For indo the laser needs to be in the uv range and we do not have it on our Aria. Indo is pH sensitive so you would have to do a SNARF-AM for detecting pH changes. We have not checked if BTC is also pH dependent.

The best measurements are 3 minutes baseline, add your substance and then measure 3-5 minutes depending on the length of the response.Make sure to choose "append data" when you reload the tube, so all the data is in one file.

The last treatment should be an ionophore like ionomycin that can punch holes in the cells so you can see if the cells responded by giving a fairly large blip.


Also our old computer crashes if we do experiments for longer than 10 minutes total. May or may not be true for the new Arias

Note the reloading of the tube should be fairly quick as calcium flux responses are very quick (microscopy is much better at this measurement).

The protocol for dye loading is pretty much what I used when working in microscopy.
Dopamine D2 receptors regulate in vitro melanotrope L-type Ca2+ channel activity via c-fos.
Endocrinology. 1995 Feb;136(2):614-21.


We would be happy to publish the ratiometric methods we use if such experiments in flow cytometry have not been detailed already.
Hope that helps!
Parul





-----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 10:59 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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