Colleagues: In the interests of making this a good learning experience, let me expand on this issue of naming parameters as FL1, FL2, FL3 etc If there were only one instrument on the market, and there were only 3 fluorescent probes, and you could only measure them in set places, the argument that these could stand might be possible, but it would still be weak. The facts are, that there are many instruments, many possible fluorochromes and a heap of possible combinations. Just because one particular benchtop instrument, which is frequently referenced may be restricted in its filter set for FL1, FL2 and FL3, is no excuse or reason to allow such use of a terminology. Take the experiment that started this discussion (this time!). How are people who don't have this particular commercial machine to interpret the question or answers? If the information is not correctly formatted into scientifically supported jargon, many people would not be able to interpret it. This is why we don't accept RPMs for wash speeds. We demand that users give us RCF (g) values - which are interpretable across all centrifuges. RPM can also give you the correct information, but only if you list the centrifuge Model, the head model and the type of buckets and tubes...... then it's only useful if you have that centrifuge, head and bucket! Otherwise, you have absolutely no clue what the wash conditions are! Bottom line, is publications do not accept RPM. So same goes for flow. If you want to discuss fluorescence signals, it is appropriate to use a terminology that is interpretable in a scientific manner. Here are my suggestions, and these are the ones that we require for those who contribute to Current Protocols in Cytometry, the manual that I hope all of you have in your labs!!! To express a fluorescence signal on any machine we request the fluorochrome (example follows), and the center of the wavelength band. e.g. FITC-525 nm, PE-575 nm. If there is an antibody attached, we would suggest it be written CD4-FITC-525 nm. Of course, there are many times when we might simply measure a frequency band, so FITC may not be appropriate. In such case it might be "green fluorescence-515 nm" or similar. If you are using a system with fixed filters that might be "green fluorescence- 525 nm". On flexible systems where all sorts of filter changes can be made, then you obviously must express the specifications of your detection in detail. If you don't know what the exact specifications are for each PMT, you shoudl find out, print them out and paste them on the front of the machine. This is not perfect, as it does not necessarily indicate the width of the band, etc. However, all of these can be stated in figure legends. If you want to indicate where on your cytometer this is measured from, you can add the PMT designation as well. e.g. CD4-FITC-523 nm-FL1 No system is perfect, but the system I have suggested is totally reproducible by any one with any machine. There are many minor modifications to the above that are perfectly acceptable. I take this issue quite seriously, and if you are unlucky enough to have your manuscripts sent to me for review, I won't accept FL1, FL2, etc. I just return the M/S and suggest that the author write in an acceptable scientific format. That might sound tough, but in fact there are few if any journals that will accept figure references that lack units. That is in fact what we constantly see in flow cytometry. We need to raise our standards to what the rest of the community requires. OK, I guess I have opened this discussion up and I am putting on my armor plated labcoat with the super-slick "slipery-kote" and will await the onslaught! Best wishes Paul Robinson On 17 Dec 2001, at 11:51, Donnenberg, Albert wrote: Paul and colleagues- With due respect to the flow wizards, I think we are being WAY to nitpicky here. The single assumption necessary to make the original Rhodamine 123 question perfectly intelligible is that the questioner is using a commercial 4-color analytical cytometer with filters as supplied by the manufacturer. For the purpose of publication, we have always thought it sufficient to state that model of the analytic cytometer used to collect the data. From there, FL1, FL2 etc. need no further interpretation. In the case of sorters, where user configured optics are more common, it is worth the ink to specify the filter configuration. Albert Donnenberg J.Paul Robinson, PhD PH:(765)4940757 Professor of Immunopharmacology Professor of Biomedical Engineering Purdue University FAX:(765)4940517 EMAIL:jpr@flowcyt.cyto.purdue.edu WEB: http://www.cyto.purdue.edu
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