Justin, Several factors can change the relationship between the indo-1 ratio and intracellular ionized calcium concentration, [Ca(2+)]i. If you suspect that this relationship is being affected by experimental conditions, then you should perform your (indo-1 ratio to [Ca(2+)]i) calibration more than once (see Carl June and Peter Rabinovich for methods to perform this calibration). In the end, all of your data is expressed as [Ca(2+)]i in units like nM, and then meaningful comparisons can be made. Good luck, Eric >Greetings, > >We are running Indo-1 for calcium flux plus PE and PE-Cy5 for surface >markers on an EPICS 753. When samples are run that have been incubated >with a drug of interest, we get a higher baseline reading for the indo-1 >ratio. If we can prove that the drug is causing the higher baseline >through increased background fluorescence in one of the indo-1 channels, is >it proper to normalize the ratio baseline by either adjusting PMT voltages >or sliding the mean ratio histograms so that the baselines match or none of >the above? Thanks. > > >------------------------------------------------ >| Justin Fishbaugh | >| University of Iowa | >| Flow Cytometry Facility | >| 48 EMRB | >| Iowa City, IA 52242 | >| | >| justin-fishbaugh@uiowa.edu | >| http://www.medicine.uiowa.edu/flowcytometry/ | >------------------------------------------------ /\/\/\_ Eric Van Buren, aa9080@wayne.edu \ \ \ Karmanos Cancer Institute and Immunology & Microbiology \_^_/ Wayne State University, Detroit, Michigan, USA
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