[Cytometry] Loss of FITC signal during sorting

Geoffrey Osborne g.osborne at uq.edu.au
Sun Feb 9 18:58:33 EST 2014


HI,
Just as a side point here it might be worth check the pH of the sheath
fluid in the sorter, and sample tube. As you are probably aware FITC is pH
dependant and from memory is something like twice as bright at pH 7.5 than
pH 7. Back in the day this was widely utilised
http://www.ncbi.nlm.nih.gov/pubmed/6198189

If the pH in the sample tube is borderline to low and you start pressuring
it with a non inert gas "interesting" things may start to happen to your
FITC signal.

But then again I could be completely wrongŠ.
Geoff 
-----------------------------------------------------------
Geoffrey W. Osborne
Director of Flow Cytometry,
The Queensland Brain Institute / The Australian Institute for
Bioengineering and Nanotechnology,
The University of Queensland, St. Lucia, QLD 4072. Australia
Ph (61) 07 33466397
-----------------------------------------------------------









On 30/01/14 7:19 PM, "Berislav Bosnjak" <berislavbosnjakster at gmail.com>
wrote:

>Hi all,
>
>We are trying to sort a subpopulation of CD4+ cells labelled with
>FITC-stained Ab specific to our marker of interest. However, when we try
>to
>sort the cells on Aristos, population of FITC+ cells is lost: initially we
>get >5% of labelled cells (as on Fortessa), but population gradually
>disappears and drops down to less <2% of CD4+ cells.
>
>We have tested staining panel on LSR Fortessa and it proved to be specific
>and stable (5-6% of cells are FITC+ irrespectively on the acquisition
>time). We have checked the Aristos using FITC-labeled beads, no loss in
>the
>signal there.
>
>I would appreciate any ideas why this happens and what we could do to sort
>the cells.
>
>Berislav
>
>----
>Berislav Bosnjak, MSc
>Medical University of Vienna
>Department of Dermatology
>Experimental Allergy
>4P9.10
>Währinger Gürtel 18-20
>A-1090, Vienna
>Austria




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