[Cytometry] Loss of FITC signal during sorting

Berislav Bosnjak berislavbosnjakster at gmail.com
Thu Feb 6 08:55:24 EST 2014


Hi Robert,

Thank you very much for this very insightful comment.

In fact, we have re-analyzed samples yesterday on Aria and the FITC-stained
population of our memory CD4 T cells seems to be stable on that instrument.
We do not know at the moment is it because of the instrument itself or the
buffers.

I wish to thank you all who helped with suggestions and comments!

Cheers,

Berislav


Berislav


On Mon, Feb 3, 2014 at 12:28 AM, Robert Wadley <rbwadley at hotmail.com> wrote:

> Dear Berislav,
>
> 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.
>
> Regards
>
> Robert Wadley
> Cytometry Manager
> Mater Research
> Brisbane, Australia
>
> 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
>
> Hi,
>
> 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
> problem.
>
> 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
>
> the sort.
>
> Kind regards,
>
> Berislav
>
> ----
> Berislav Bosnjak, MSc
> Medical University of Vienna
> Department of Dermatology
> Experimental Allergy
> 4P9.10
> Währinger Gürtel 18-20
> A-1090, Vienna
>
> Austria
>
>
> 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?
> > Gerhard
> > 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
> > 650-232-5437
>
> > 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
> >
> > ----
> > Berislav Bosnjak, MSc
> > Medical University of Vienna
> > Department of Dermatology
> > Experimental Allergy
> > 4P9.10
> > Währinger Gürtel 18-20
> > A-1090, Vienna
>
> > Austria
> >
> > _______________________________________________
> > Cytometry mailing list
> > Cytometry at lists.purdue.edu
> > https://lists.purdue.edu/mailman/listinfo/cytometry
>
> > Search the list archive at  http://tinyurl.com/cytometry
> >
> >
>
>
> _______________________________________________ Cytometry mailing list
> Cytometry at lists.purdue.edu
> https://lists.purdue.edu/mailman/listinfo/cytometry Search the list
> archive at http://tinyurl.com/cytometry
>


More information about the Cytometry mailing list