Re: missing emails

Hi Gerhard,

I have noticed the same problem as you - I missed the original email of the
FL6, 9 discussion but got all the answers and comments! Sometimes I get
replies BEFORE I receive the question, although still on the same day. I put
that down to the time difference and being in Europe, as most of the emails
come from the USA. I do check spam filters and junk boxes as well and they
do not go there. So, I agree there is a mystery somewhere!

Anne



Dr Anne Wilson
Associate Investigator
Developmental Immunology Laboratory
Ludwig Institute for Cancer Research
Lausanne Branch
Chemin des Boveresses 155
CH 1066 Epalinges
Switzerland

tel +41 21 692 5971
fax +41 21 692 5995

PLEASE NOTE MY NEW EMAIL ADDRESS EFFECTIVE 1 JANUARY 2008

Anne.Wilson@licr.unil.ch



On 3/26/08 1:34 PM, "Nebe-Von-Caron, G" <g.nebe-von-caron@spdspark.com>
wrote:

> Hi everyone
> 
> I recently noticed the list contained answers to subjects of which I did
> not have the original message. I have plenty of FL-6, 9 and bay
> biosciences... but for example not did not receive the original post
> from Helen about the FACSAria shifting signals. The other recent message
> I did not get was
> 
> From: Rudjer Novak [mailto:rnovak@pharma.hr]
> Sent: Fri 3/21/2008 4:03 AM
> To: cyto-inbox
> Subject: phagocytosis
> dear flowers,
> 
> They are not blocked by the spam filter and also not sorted out by
> internal filtering.
> 
> Now it is obvious if you miss the original post as you have only the
> RE:~ messages, but I do not know how many answers might be missing as
> well.
> 
> Anybody else noticed something similar?
> 
> Gerhard Nebe-von-Caron
> Research Scientist and Biomedical Engineer
> SPD-Spark
> Swiss Precision Diagnostics
> Priory Business Park
> Bedford, MK44 3UP, UK
> Tel +44(0)1234-835474
> Fax +44(0)1234-835002
> mailto:g.nebe-von-caron@spdspark.com
> -----Original Message-----
> From: Helen Ferry [mailto:helen.ferry@imm.ox.ac.uk]
> Sent: Thursday, March 20, 2008 5:37 AM
> To: cyto-inbox
> Subject: FACSAria shifting signals
> 
> 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
> 
> 
> 
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