>Dear Flow-ers, > I have a colleague who wishes to analyze and sort cells that bear GFP >and CFP (cyan fluor. protein) reporter constructs. >Has anyone had experience doing this type of analysis with a Vantage SE >and an Enterprise II laser (6 color config). I realize that using a 488 >nm line is suboptimal for CFP, but is it feasible? CFP has a few % excitation efficiency at 488 nm. However, since its emission peak is at 475 nm, it is a poor choice to use in conjunction 488 nm excitation. It is possible to get usable signal with shorter excitation, 407 nm from a krypton or 457 nm from an argon. For the 4o7 nm excitation a 440/40 ish bandpass works OK but is not optimal. For the 457 nm excitation, there is only a narrow band below 488 nm to work with. If you can get UV out of your laser, it may be a more feasible alternative to 488 nm. A very functional combination using standard argon lasers is to excite CFP at 457 nm and YFP at 514 nm. It appears that your user may be trying to move an application developed for microscopy to flow without taking into account the differences in available excitations between the two technologies, a problem I have encountered several times. Marty > >Any info would be very much appreciated. > >Thanks, > >---------------------- >Andrew Herman >Department of Pathology & Microbiology >University of Bristol >University Walk >Bristol BS8 1TD >Tel. +44 117 928 7511 >Fax. +44 117 928 7896 >A.Herman@bristol.ac.uk -- Marty Bigos Director, Flow Core Gladstone Institute of Virology and Immunology Building 3 SFGH Rm 509 415-695-3832
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