[Cytometry] Cytometry] seeking advice with measuring Calcium using Fura-3 and Fluo3 or 4?

Gary Warnes g.warnes at qmul.ac.uk
Wed Feb 4 04:08:55 EST 2009

Hi Andy,

      I have a protocol on my website for Fluo-4, see link
http://www.icms.qmul.ac.uk/flowcytometry/protocols/Calcium/Fluos.htm  I
normally use Indo-1 instead of Fura-2 on flow cytometers as this excites
with UV and emits at about 400nm and over 500nm enabling a ratiometric
measurement see the link


Fura-2 is normally used on microscopes as this dye is excited with two
wavelengths which are between 300 & 400nm so you could try using UV and the
405nm line to excite the Fura-2 (could not find Fura-3 in the Molecular
probes hand book) but I'm not sure this would work as nicely as Indo-1 does.
Are you comparing the FACS calcium with imaging with Fura-2 I was asked to
do this once but then my confocal is set up to run Indo-1 so we switched


I normally do my Calcium experiments at RT as they work fine in most cases
if you need to use 37C then I put a small beaker of 37C water on the probe
at the same time as the tube and just replace the water after every run.
This approach is a bit difficult after you have added the agonist to the
tube and replacing the tube quickly on the machine so you might want to use
the Vantage setup.


Best of luck with this experiment 


Dr Gary Warnes

Flow Cytometry Manager 

Flow Cytometry Core Facility

Institute of Cell & Molecular Science

Barts and The London School of Medicine and Dentistry

London University

4 Newark Street 


E1 2AT

Tel No.0207-882-2402

Fax No. 0207-882-2180



Dear flowers,

    I was wondering if someone could help me with a protocol for measuring
intracellular Ca++ in murine T cells using the probes Fura-3 and Fluo-4.

I didn't find more than a couple of citations of the 2006 paper by Sheree
Bailey and MacArdle, and I was wondering if this assay is routinely being
used on a FACS Canto II or LSR II. Is there an advantage to using a FACS
Vantage's temperature controlled sample holder instead of the benchtop


Many thanks,




Andrew Herman, Ph.D.

University of Bristol

Department of Cellular & Molecular Medicine Director, Flow Cytometry
Facility School of Medical Sciences University Walk Bristol BS8 1TD UK



Mobile: 079 6782 9670


A.Herman at bristol.ac.uk




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