[Cytometry] Loss of FITC signal during sorting
rbwadley at hotmail.com
Sun Feb 2 18:28:58 EST 2014
One other point, there is a big difference between your Fortessa and your Astrios.
The Fortessa analyses your cells as they move through a cuvette (like perhaps 99.9% of all analysers on the market), directly attached to the cuvette is the lens for the collection optics, so you collect a much greater % of the fluorescence from the FITC molecules.
The Astrios is a stream-in-air sorter, the lasers intercept the cells in air and there is a comparatively large distance from there to the collection optics, so a smaller % of the fluorescence from the FITC molecules is collected.
FITC is a relatively dim molecule at the best of times, stream-in-air sorters try to make up the difference with more powerful lasers, but sometimes this is not enough. In some cases dye choice will directly influence the sorter most useful to your experiment. I got caught out very badly last year with researchers using PerCP-Cy5.5 on CD19 which looks beautiful on all my analysers, but is practically invisible on the Astrios, so I was restricted to immensely long sorts on my Aria IIu, or the researchers had to shift CD19 to APC (which they could have done had they continued).
I am discovering lots of interesting anomalies sorting on a multi-laser, multi-colour Astrios, but doing the prep (and if I can't stop them) the post sort analysis on a conventional analyser. This is not a problem with the Astrios, it is an education process for the researchers. It was different when I only had the MoFlo MLS for analysis & sorting, because you were forced to make the right dye choices right from the beginning. Analysis on a cuvette machine, when prepping for a sort should be followed by an initial analysis on the sorter, before you make your final dye selections, so that the sort works the way the researcher intends.
From: berislavbosnjakster at gmail.com
Date: Sat, 1 Feb 2014 22:02:56 +0100
To: g.nebe-von-caron at alere.com
CC: Cytometry at lists.purdue.edu
Subject: Re: [Cytometry] Loss of FITC signal during sorting
Thank you for your suggestions, they are very valuable to us.
We are trying to sort out a subpopulation of CD4+ T cells which are
positive for our marker. The only Ab currently we have for the marker is
FITC labeled, so we have to stick with it. It appears to be a FITC-related
We will try to change the sorting instrument and thus change the sheath
fluid, hopefully this will help. We will also try to cool the sample during
Berislav Bosnjak, MSc
Medical University of Vienna
Department of Dermatology
Währinger Gürtel 18-20
On Sat, Feb 1, 2014 at 3:14 AM, Nebe-Von-Caron, G <
g.nebe-von-caron at alere.com> wrote:
> You don't say what sample you r using and what counter stains. If you look
> at monocytes they could adhere to the tube if it's cd3 specific it could be
> capping. Check where the cells are in sides scatter
> Also is it a Fitc or cd4 problem?
> Sent from my my phone:-)
> On 31 Jan 2014, at 23:10, "Rupak Neupane" <rneupane at cellerant.com> wrote:
> Did you keep the sort sample at 4 degree? I have seen the same problem on
> one of my Aria and realized that the sample chamber cooling was broken. The
> sample on the other Aria was fine for long sort and once we fixed that, it
> was fine again. Hope that Helps.
> Rupak Neupane
> Manger, Flow Cytometry Core Facility
> Cellerant Therapeutics, Inc.
> 1531 Industrial Rd
> San Carlos, CA 94070
> rneupane at cellerant.com
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu [
> mailto:cytometry-bounces at lists.purdue.edu<cytometry-bounces at lists.purdue.edu>]
> On Behalf Of Berislav Bosnjak
> Sent: Thursday, January 30, 2014 1:20 AM
> To: Cytometry List
> Subject: [Cytometry] Loss of FITC signal during sorting
> 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 Bosnjak, MSc
> Medical University of Vienna
> Department of Dermatology
> Experimental Allergy
> Währinger Gürtel 18-20
> A-1090, Vienna
> Cytometry mailing list
> Cytometry at lists.purdue.edu
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