Maciej, >From what you've listed, there's no reason for the "autofluorescence" that you describe . . . something else is going on. Time for some detective work. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Flow Core 7416 CCGC 0946 (734) 647-3216, fax (734) 936-7376 kukuru@umich.edu >>> Maciej Simm <simmmmer@yahoo.com> 02/13/01 05:15PM >>> Greetings, An investigator looking to establish a staining protocol for annexin 2 has been bringing cell lines to look at nonspecific isotype matched binding as well as the FITC mAB for annexin2. the unstained cells fluoresced in the fourth (!!) log on FL1, FL2 and FL3 with the following instrument settings: FSC voltage E-1, 8.0, linear SSC voltage 499, 1.0, linear FL1 voltage 640, 1.0, log FL2 voltage 641, 1.0, log FL3 voltage 650, 1.0, log threshold = 52, FSC as primary (population scatter plot looks ok) comensation values - FL1 - 1.0 % FL2 FL2 - 20% FL1 FL2 - 1.0 % FL3 FL3 - 20% FL2 Needless to say, we didn't get much sensical data out of the isotype control or the annexin II. The cell lines which had this problem are: Hl-60 8226 OPM-2 KMSII Raji I have NO idea how to quench this level of autofluorescence, I have never seen it this strong! We tried incubating in 50mM NH4Cl in PBS but we saw no difference. If anyone here has any experience with this sort of problem, please let me know. Thanks in advance, Maciej __________________________________________________ Do You Yahoo!? Get personalized email addresses from Yahoo! Mail - only $35 a year! http://personal.mail.yahoo.com/
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