[Cytometry] Loss of FITC signal during sorting

Berislav Bosnjak berislavbosnjakster at gmail.com
Sat Feb 1 16:02:56 EST 2014


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
>
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