Re: FACSAria shifting signals

Hi Helen.

With which combinations of fluorochromes are you having this problem? What do
you have in the PE channel, if you exclude the PE antibody does it still occur?
Have you looked at the compensation between the PE channel and the offending
channels when you are seeing this curious effect? I don't really have an
explanation I'm afraid but I think you'll have to identify which of your
reageants is causing the effect and swap it for something else. You could try
turning off all the lights I suppose, maybe turning off the Accusort laser,
stray red light might cause such a problem.
Just a couple of ideas.
Best of luck

Simon


LQuoting Helen Ferry <helen.ferry@imm.ox.ac.uk>:

> Dear All
>
> Sorry, this is a long one.......
>
> I am hoping that there is someone out there who has experienced the same
> problem we are experiencing with our SORP FACSAria, and more importantly can
> offer a suggestion for how to solve it.
>
> For months now, we have seen signals increasing in the PE-Cy5, PerCP-Cy5.5 or
> PE-TxR channels; all are off a 150mW green laser. To describe it more
> precisely, the negative/low cells become positive whereas we detect little/no
> increase in the positive population. The signals return to normal when the
> sample is repeatedly unloaded, then run again.
>
> In most instances, although not all, we have seen the signals shift after
> switching from bulk to single cell sorting into plates. (We use 488 alignflow
> beads from Invitrogen to confirm the single-sorting is working correctly i.e.
> one bead/well.) We have also seen the problem come back after the single-cell
> depositor was switched off when we swapped back to bulk sorting. We have run
> the same samples on our LSRII but we have never observed the same effect.
>
> We had frequent discussions with BD, who believed that the problem was due to
> 7-AAD saturating the flow cell then staining all the cells non-specifically;
> even though it is not possible to replicate the effect by overloading the
> system with 7-AAD. We agreed to stop using 7-AAD and swapped to DAPI, however
> the problem persisted.
>
> After transposing DAQ boards and PMTS etc, BD decided to exchange the
> instruments which we all hoped would be an end to the matter. However last
> week during a single-cell sort we observed the negative/low cells in
> PerCP-Cy5.5 and PE-TxR channels increase just as before. 7-AAD has never been
> used with this instrument and we are still using DAPI.
>
> BD are now convinced that it is a user/application error, but are still
> unable to offer an explanation as to what could cause such a phenomenon. We
> concede that the problem may indeed be related to the application, possibly
> in combination with the Aria, however we would prefer to prove this
> conclusively so that we can eliminate the cause.
>
> BD suggest that we now stop using viability dyes completely. However as we
> are trying to sort rare populations we would prefer not to do this, as it is
> well-documented that Abs bind non-specifically to non-viable cells,
> significantly increasing the background.
>
> We are now at our wits-end as it is really affecting our ability to perform
> sorts and therefore downstream experiments, not to mention the wasted time
> and money when the sorts fail.
>
> Has anybody out there seen the same thing or offer a suggestion of what we
> could try to remedy this? Any help would be really appreciated.
>
>
> Best wishes
> Helen
>
> Helen Ferry, D.Phil
> FACS Manager
> Haemopoietic Stem Cell Laboratory
> Weatherall Institute for Molecular Medicine
> John Radcliffe Hospital
> University of Oxford
> Headington
> Oxford
> OX3 9DS
> United Kingdom
>
> +44 1865 222340
>
>
>


-- 
Simon Monard
Cytometry Lab Manager
Rua do Campo Alegre, 823,
4150-180 Porto - Portugal
Tel +351 226 074 900 ext 3102 . Fax +351 226 099 157
Cellphone # 919 036 680

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