Mini Tutorial on use of FL1, FL2 etc
Fischer, Randy (NIAMS)
fischer1 at mail.nih.gov
Wed Dec 19 08:49:52 EST 2001
No need for the armor plating, yet. Actually Paul, when I initially read
your first response, I did think it a little harsh. Then I tried as a test
to myself to answer the e-mail in question and found I needed exactly the
information you mention below. I believe that I am guilty, like many others
on this list, of being maybe a little lenient with many of my colleagues
simply because I do get tired of correcting them every time they say FL1 or
FL2 instead of FITC or PE or whatever they are using. And with the machines
where the light path can be changed so FITC is now in FL3 etc, this does
become a serious issue for those wishing to reproduce or build upon previous
data. As you have often pointed out in the past, education may be the best
tool for preventing this problem. Anyone wishing to use our facility is
given at least a verbal test to find out what they know about Flow and its
relative power as an analysis tool. After that, we tailor their use levels
and further education to their experiments.
Happy Holidays to all,
Randy T. Fischer
Building 10, Room 6D57
9000 Rockville Pike
Bethesda, MD 20892
fischer1 at mail.nih.gov
> From: J.Paul Robinson
> Reply To: jpr at flowcyt.cyto.purdue.edu
> Sent: Monday, December 17, 2001 4:30 PM
> To: Cytometry Mailing List
> Subject: Mini Tutorial on use of FL1, FL2 etc
> 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
> probes, and you could only measure them in set places, the argument that
> 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
> 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
> 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
> (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
> 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
> perfectly acceptable.
> I take this issue quite seriously, and if you are unlucky enough to have
> 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
> 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
> here. The single assumption necessary to make the original Rhodamine 123
> question perfectly intelligible is that the questioner is using a
> 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 at flowcyt.cyto.purdue.edu
> WEB: http://www.cyto.purdue.edu
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