Dear Andrea Just to state that I do not want to insult any one and any manufacturer and try not to come over as arrogant....(how many disclaimers should I try?) In line with Mario Roedere's recent comments, your question should not arise, as clearly you must have a local flow jock or core manager that should help you to set up your instrument correctly and check those problems out, in particular as you seem to have access to several instruments you clearly must have the local expertise to solve such problem. I hope this will stir a few more thoughts on the problem we try to tackle with the data presentation and -far more important- data interpretation. As complex as a cytometer is, you can drive it without a licence. If cytometry is used for "research only" it's failed application does not necessary endanger human life directly (ignoring Mario's blood pressure). Also I do not want to drive in a formula1 race or fly a jumbo--- but that diverts too far. I find myself quite often these days asking people to contact their local experts, in particular if their questions show a lack of knowledge on basic principles of cytometry or the complexity of the problem is bigger than what can be said in a couple of lines. If the people are meant to be the local experts themselves, then they should consider careful reading and going on a good flow course to ensure they get the basics as this mailing list (probably the must helpful and informative mailing list on the web) does not replace a fundamental training in cytometry. Perhaps as peer reviewers as much as much as members of this list we should sometimes speak out such recommendations, but better in private than publicly ridiculing someone else. Now in this case Andrea has shown good observatory skills and a valid scientific approach of comparing measurements in different instruments. From here I can neither comment on the state on the instruments, the amplifier voltage settings, the use of signal off-sets, filter combinations etc.pp.. However, a lot of us will be tempted to try and resolve the problem - as we are a very helpful bunch and probably all been stuck in similar situations ourselves before?. The fact that she contacts the mailing list indicates to me that it is sometimes easier to get local access to another instrument than local technical expertise. Now that problem will not be solved by discussing data presentation in published papers and is probably what the old-timers (I think I am more a TOG judging by the accumulation of senior moments) try to indicate in their approach. As Howard said quite rightly, in most cases bad flow data do not put the conclusion of a paper in question. However, if that is the case, then most papers have a good tradition of publishing letters to the editor in order to rectify such incidents. Regards Gerhard -----Original Message----- From: Andrea Dewar [SMTP:andrea.dewar@imvs.sa.gov.au] Sent: Thursday, November 15, 2001 12:58 AM To: Cytometry Mailing List Subject: CFSE Compensation Problem Hi there. I'm having a big problem with compensating cells double stained with CFSE (FITC) and PE on our Coulter flow cytometer. On a dot-plot, to bring single stained CFSE cells into the 1st decade of the scale, 75% or more of my cells slam against the axis before the remaining 25% of cells are pulled into the first decade region. When my cells are counter stained with a PE antibody, the majority of negative cells are still slammed onto the axis, so that if you don't look closely, you can't see these cells and all events appear to be double positive (whereas at least 25% of the cells are actually negative). This is a problem specific to the Coulter machine as the same cells were run on a BD machine. The cells did not slam into the axis when compensated on the BD machine. The level of compensation used on the Coulter machine is not high (28.1) and the problem is not becuase the cells are stained too brightly (the problem still occurs when the cells are sitting at 2000). Any suggestions would be thoroughly appreciated. Andrea Dewar PhD Student Institute of Medical and Veterinary Science Adelaide South Australia AUSTRALIA
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:40 EST