Janet -- The reason people use fluorescent area and fluorescence pulse width measurements is to try to remove doublets, triplets, etc. from their data prior to mathematical analysis for cell cycle information. Some instruments have the ability to make some or all of these measurements and some don't. Beyond that, some instruments make relatively crude measurements while others have considerably higher resolution. So whether you use a "hardware" approach using these electronic processing methods, or a software that corrects for doublets based on triplets, or other algorithms, depends on what instrument you have, what application you are running (including what the relative size of your cells compared to the laser beam width which is an important factor, or if you have high-resolution, real-time laser beam subtraction capability which greatly changes the problem). Your question really has a number of factors that make it difficult to give a very brief answer. I could, but it might be wrong for your (or another ListServ reader) circumstances. So rather than either giving a simplistic answer or, on the other extreme, a very lengthy answer with some of the nuances, I will refer you to a paper that an NIH group and I published last year. We not only discuss your question but explain why you get different quality of results on different instruments using these hardware approaches on the signals as they come in or alternative software approaches on the data after acquisition. So I hope you find this article helpful and informative. If not come back for more...I've been doing these measurements on a wide variety of cell types for more than 20 years and have learned much in the school of hard knocks! Wersto, R.P., Chrest, F.J., Leary, J.F., Morris, C., Stetler-Stevenson, M., Gabrielson, E. "Doublet Discrimination in DNA Cell Cycle Analysis" Cytometry 46(5): 296-306, 2001. - -Jim Leary James F. Leary, Ph.D. Chief, Molecular Cytometry Unit, Div. Infectious Diseases Professor of Internal Medicine, Pathology, Microbiology & Immunology, Biophysics, Human Biological Chemistry & Genetics, and Biomedical Engineering 4.216 Mary Moody Northen Pavilion - Route 0435 University of Texas Medical Branch 301 University Blvd. Galveston, Texas 77555-0435 Tel: 409-747-0547; Fax: 409-747-0550 -----Original Message----- From: janet dow [mailto:jldow@unity.ncsu.edu] Sent: Monday, January 07, 2002 12:41 PM To: cyto-inbox Subject: meaning of FL2-area and FL-2 width I know this is going to sound like a stupid questions but we(me and several post docs) are currently involved in a heated debate as to the nature and meaning of FL2-area and FL2-width in connection with the measurement of DNA content(PI stained) of ethanol fixed cat lymphocytes. Can someone give us a good definition of each and an explanation of why we use these for DNA measurement instead of FL2-Height? We are using a BD FACSCalibur for our work. I have done DNA work for years and I have never really thought about this before-this is how I was taught to do it. Again I am sure this is result of never having taken a formal course on flow and just picking it up from the person who previously held my job and other investigators. I have searched the archive and can not find a good explanation. Maybe I need a course in using the search engine for the archive!!! Thanks in advance. Janet Dow Janet Dow Research Technician and Manager Flow Cytometry Facility North Carolina State College of Veterinary Medicine Room C-314 Raleigh, NC 27606 (919)513-6364
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