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 ********************************************************************** This e-mail transmission may contain confidential or legally privileged information from ICON Central Laboratories, Inc. intended only for the use of the individual(s) or entity named on the transmission sheet. If you are not the intended recipient, you are here by notified that any disclosure, copying, distribution, use or taking of action in reliance upon the contents of this e-mail is strictly prohibited. If you have received this e-mail transmission in error, please notify us immediately by telephone or e-mail so that ICON Central Laboratories, Inc. can arrange for the return of the documents to us at no cost to you, and then please delete the message. Thank You, ICON Legal Department, ICON Central Laboratories, Inc. (631) 777-8833 **********************************************************************Received on Wed Mar 26 15:18:00 2008
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