Date: Fri Feb 08 2008 - 09:42:02 EST
I may repeat what others already stated: antibody capturing compensation beads (BD or others) are the key in most cases for the proper compensation. We have links to the BD beads on our intranet site and tell every new user about them. Take a little time to explain to them how they work, how reliable they are and what the pitfalls of not employing the beads are. We also run multi color samples on bi-exponential plots on the Diva machines. Remember: in Diva you can tweak your automatically compensated samples (not that you need to do it with proper controls - but occasionally we had to exploit Diva's flexibility). Also (in Diva) you can change the compensation weeks later if a mistake was made. Akos __________________________ Akos Szilvasi NIBRI Core Laboratory Services manager USCA, 601-5301 Novartis Institutes for BioMedical Research, Inc. 100 Technology Square Cambridge, MA 02139 USA Phone: +1 617 8717177 Email : akos.szilvasi@novartis.com Felix Heymann <felix.reichel@ukb.uni-bonn.de> 02/07/2008 07:24 PM To Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> cc Subject Re: Manual vs. Auto compensation Hi Nidal, we use to have frequent discussions about compensation issues at our institute (as probably all flow based facilities do ;) ) and I will just pass on some thoughts I picked up during my time here. First of all, from personal experience, autocomp works just fine. I just recently compared the two (manual and auto) when teaching one of our younger technicians how to use our FACS Canto and we therefore did it the quick way and the 'proper' way and guess what.....we ended up with almost the same compensation values. So, either way, if you do it properly, it should work. The manual guys are normally the ones having started flow cytometry in the old FACSScan or Calibur days and refuse to 'let go', but the thing is, even when you mess up your compensation on the new, digital machines, you can still work on that AFTER your measurement. Much more important is the setting of the PMT-voltages, this is the only thing you have to make sure that your signal to noise ratio is ok with your settings. Some people at our lab don't even bother compensating the samples while measuring and just take the single stains over to their FlowJo analysis and do the compensation there. That's sort of autocompensation, too, since it's the same matrix based system as with the DIVA software (I don't know about the underlying algorithms, though). To see whether you have over or undercompensated your sample, you could use the biexponential display, which gives you an idea whether you have produced 'negative' cells when overcompensating the signals. Just makes plots prettier though, since your cells won't all sit on the axis, but give you a nice cloud instead. The thing is, that plots may look different when you change your compensation procedures (and since people just agree to what they are used to, different may be a 'bad' thing ;) ), but as long as you can make out your population, it should be fine. Only problem of course would be if you look at expression strength of markers, since it might be difficult then to find out what is high expression, low expression or no expression at all, that's something you would have to establish for the specific scientific question you want to answer. In terms of using different cells for compensation, you are right when it comes to the issue of autofluorescence....BUT....the difficulty may be, that in your sample you just don't know if your specific markers really are detectable and (if not) may leave you with no clear positive staining whatsoever or cell fractions so small that you just can not use them for compensation. We had the discussion whether it is ok to switch to a different marker then labeled with the same colour (eg using CD8 FITC instead of CD86 FITC) and this should be ok at least with the non-tandem dyes (I will come to that issue in a second). All you have to make sure, that you could detect both FITC-signals with the same PMT-Setting, the spillover should be the same then and you get a nice and clean positive signal when using a strong marker. I would rather (as you stated) use my tissue sample and switch my Abs rather than sticking with my markers I use for the analysis and take a different tissue (eg spleen instead of liver). That rules out the autofluorescence issue. Only problem with switching markers is the use of tandem dyes, simply because the FRET-efficiency depends on the quality of the conjugation and the amount of dye degradation (which is unavoidable after some time). Old tandem conjugates can be particularly nasty when using them together with the single dye (eg PE together with PE-Cy7). The degradation of the PE-Cy7 would leave you with a perfect PE-signal and that would be impossible to discreminate from your PE-labled Ab. So it's quite useful to check your tandem dyes from time to time (and keep them dark and cold as much as possible). Hope that helps, that's as far as I've gotten through the jungle of the flow-cyto-how-to's regards Felix Felix Heymann Institute for molecular medicine and experimental immunology University clinic Bonn, Germany > Hope all are having a great weekend and not in the lab on a Saturday > night. > > I'm a relatively knew guy to the sort/flow world and need to set some > things straight so I hope those with more experience can give me their > feedback. > > I have people in the lab who complain about me not doing manual > compensations and instead have the Aria calculate them for me. And > when I have the Aria compensate, they want to manually compensate > after they record the data because the plots "don't look right." I > mean, isn't that simply producing "make-belief" data? Or am I missing > something? I'm not trying to criticize those who do manual comp. I > just want to understand why do I need to manually compensate when it's > all based on mathematics and can be done by the machine? > > And the frustrating part is that when they bring me the compensation > samples, they contain cells that are different from the ones in their > analysis samples. I try to tell them that the autofluorescence of both > cell types is most probably different and that might be the reason why > some of the compensations don't turn out correctly. But they dismiss > my suspicion, claiming that it should not affect the compensation > since we're dealing with colors and not cell density. Is that true? > > I'm stressed out. I need some ice cream. > > -- > Nidal Muvarak > Abramson Research Center > Children's Hospital of Philadelphia > 3615 Civic Center Blvd > Philadelphia, PA 19104 _________________________ CONFIDENTIALITY NOTICE The information contained in this e-mail message is intended only for the exclusive use of the individual or entity named above and may contain information that is privileged, confidential or exempt from disclosure under applicable law. If the reader of this message is not the intended recipient, or the employee or agent responsible for delivery of the message to the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please notify the sender immediately by e-mail and delete the material from any computer. Thank you.
