Beth, We use a 3-color FacsCalibur with a 488 nm laser. I use Fluo3/Fura Red or (better) Fluo4/Fura Red to measure calcium mobilization in B cells (J. Immunol. 162:2717). I also helped another lab here use Fluo3/Fura Red in human PMNs with very good results. Fluo3 alone is less sensitive and less consistent than Fluo3+ Fura Red as a calcium indicator. Computing the ratio of fluorescence from 2 dyes allows you to correct for cell to cell variability in dye loading. Good luck Lynn Dustin >Hello everyone, > >I have just started a new project measuring the change in intracellular >calcium >in neutrophils using flow cytometry. No one in our lab has done this >before so I >just started using fluo-3 because we had some. In my readings I am lead to >believe that a ratiometric approach using fluo-3/fura red is a better way >to go. I >have done a literature search and have only found one reference by Novak and >Rabinovitch in 1994 describing the method. > >I would like to know: > > 1. Which method people are currently using for a bench top analyzer > with a 488nm argon laser? > > 2. Is anyone really using the fluo-3/fura red method? > > 3. Is this method better than just using fluo-3 alone? > >Your input will be greatly appreciated. Lynn B. Dustin, Ph.D. Assistant Professor Molecular Microbiology and Immunology St. Louis University School of Medicine 1402 S. Grand Blvd. St. Louis, MO 63104 phone: 314-577-8415 or -8420 fax: 314-773-3403
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