RE: FL-6 issues - a nonsensical label!!

I encourage all operators to take full advantage of the parameter naming
functionality in the instrument software.  Not only does it help the
investigators get used to using "real" labels for their parameters, but
it makes recalling/printing data much more informative.

For example, for "FL1" on a standard configuration MoFlo (488 nm laser,
530/40 filter), I name the parameter "Fluorescein CD4 [530/40 100mW
488]" or "eGFP [510/20 150mW 488]".  It makes publication of images much
easier and informative, and pastes nicely into a laboratory notebook.

This way, printouts of the histograms/dot plots will have the key
information on the axis, and I don't have to refer to notes/notebook to
recall the specifics.

Andrew

Andrew Beernink
Senior Applications Specialist
Beckman Coulter, Inc.
Fort Collins, Colorado
(858) 353-7007
(858) 435-1137 efax


-----Original Message-----
From: J. Paul Robinson [mailto:jpr@flowcyt.cyto.purdue.edu] 
Sent: Saturday, March 15, 2008 6:43 PM
To: cyto-inbox
Subject: Re: FL-6 issues - a nonsensical label!!

Well I nearly went ballistic when I read this too ....and I just left
it.......but since Howard opened the door.....

About 13 or 14 years ago, any  manuscript that was sent to me for review
that used figures with FL1, FL2. etc. was returned to the editor
unreviewed and identified as not acceptable for publication. Now an
associate editor of Cytometry, I won't even send a paper out for review
if the author uses nonsensical labels. There is no place for a 
discussion of FL1, fl5 OR fl15...	

We banned the use of these label in Current Protocols in Cytometry when
we started CPC in 1996.

Cytometry is a science - as such, we should expect carefully documented
technical notes. That includes references to the nature of the signal
being described.

I suppose it is important for is to constantly remind people of these
issues, but they are not minor issues when one is trying to work out
what exactly is being reported on.

Kind regards

Paul Robinson



Howard Shapiro wrote:
> James Marvin wrote:
>> I've noticed this also.  I chalked it up to residual DAPI in the 
>> sample line for a while but im not sure thats really what it was.  
>> And your right, its didnt happen all the time, and i was running lots

>> of fixed samples so thats why i thought it was DAPI.  I think that I
>> watched the Violet 1/FL-6	fluorescence increase as the sample went

>> through, another reason i thought it was dapi.  I believe it would 
>> pop up in the FL-7/violet-2/pacific orange detector also.
>>
>> Let me know what you come up with
>>
>> thanks
>> J
>>
>> At 03:06 PM 3/11/2008, Kerry Lavender wrote:
>>> Hello Flow-ers,
>>>
>>> I wonder if anyone has ever experienced or has a solution to this 
>>> problem?
>>>
>>> We have been running 8 colour panels on a Cyan machine for about 12 
>>> months now with no problems. Suddenly, and with no particular 
>>> pattern to it we are occasionally seeing HUGE and BIZARRE 
>>> autofluorescence/non-specific staining ONLY in the FL-6 channel (and

>>> primarily affecting the negative population).
>>>
>>>  It has happened with 3 separate Pacific Blue conjugates now, 2 
>>> extracellular markers (CD3 PB and Vb11 BT+ Strep Pacific Blue) and 
>>> one intracellular marker (TNF PB). And in a number of staining 
>>> panels ranging from 4-8 colours. Sometimes it manifests as 3 
>>> populations shifted up along the PB-axis even if the sample is 
>>> negative or contains no pacific blue antibody.
>>>
>>> We have tested the possibility of it being due to old formaldehyde 
>>> containing solutions but do not see any improvement with new ones. 
>>> We have also ascertained that it is not an issue with our specific 
>>> machine as it reproduced on a second machine. There appears to be no

>>> rhyme or reason to when it occurs in regard to stimulated vs 
>>> non-stimulated samples, permeabilization, presence of protein 
>>> transport inhibitors or length of time sitting in fixation buffer.
>>>
>>> We are really at a loss - can anyone help??
> I am not suggesting a solution to the problem posed,	but pointing out

> another, potentially serious, problem.
> 
> I find it difficult to believe that, with the profusion of flow 
> cytometers with as many as five working laser beams and 18 
> fluorescence measurement channels, "FL-6" will be the same for all of 
> them. If you are measuring fluorescence, you need to specify the 
> excitation wavelength (usually easy, because it is a laser line) and 
> the emission region, defined for bandpass filters by center wavelength

> and bandwidth (e.g., 520/30) or for long or short pass filters by 
> cut-on or cut-off wavelength (e.g., 665 LP). Yes, I know, FL1, FL2, 
> and FL3 are pretty standard in instruments with 488 nm excitation, but

> the standard that we in ISAC would like to impose for publication will

> get rid of them, too, in favor of descriptions that don't require 
> familiarity with any particular kind of cytometer to understand. I 
> have run across a number of situations in which people couldn't get 
> the same results because they didn't realize they were using different

> filters for "FL4" or whatever, and the more choices we have for 
> excitation wavelengths and measurement regions, the more often this 
> will happen, unless we become more precise in our characterization of
measurement channels.
> 
> -Howard
> 


--
J. Paul Robinson
SVM Professor of Cytomics
Professor of Immunopharmacology & Biomedical Engineering Director,
Purdue University Cytometry Laboratories President, International
Society for Analytical Cytology

Purdue University Cytometry Laboratories Bindley Bioscience Center
1203 West State Street
Discovery Park, Purdue University
West Lafayette, IN 47907-2057
Ph (765) 494 0757; Fax (765) 494 0517
email: jpr@flowcyt.cyto.purdue.edu
www.cyto.purdue.edu

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