Hello, I recently posted a question about calcium measurement. I received a couple of helpful responses but received many more responses from people wanting to know what I found out. He it is: Dear Beth, We have been using the fluo-3/fura red method for a couple of years here and it works very successfully: In response to your specific questions: * We use the Coulter XL measuring Fluo 3 emission in the FL1 detector and Fura red in the FL4 detector. * Because we analyse lymphocyte activation with both antibody stimulation and using peptide pulsed B cells I trigger on FL4 thus excluding non-loaded cells from the analysis. Here is a brief method: Cells were suspended in 30mM HEPES buffered RPMI and loaded with 3mM Fluo 3-AM and 8mM Fura red-AM (Molecular Probes Europe, Netherlands) in the presence of pluronic F127 detergent (Sigma , UK, final concentration 0.05%) for 30 minutes at 37*C. Cells were then washed and re-suspended at 5x105/ml and warmed to 37*C for 10 minutes prior to use. Calcium flux was measured using a Coulter XL MCL flow cytometer (Coulter, USA). Mean ratio of Fluo 3/Fura red fluorescence was measured during the acquisition time course and expressed grapically to indicate calcium flux. * The method is better than Fluo-3 alone since a ratiometric technique renders the data independent of dye loading, cell size, dye leakage and photobleaching. The low noise and high sensitivity are comparable to indo-1 at the ranges of calcium concentrations found in T cells. Regards, Simon 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 Hi Beth, Yes the ratiometric method is much better. Using just Fluo-3 the intensity of signal is dependent on the loading efficiency, so if you are comparing several cell preps they may be all different. If you use the ratio your measurement is no longer affected by loading efficiency and also the difference between resting and full deflection will be greater as the fura-red signal gets less as your fluo gets greater. I found it works better if you use FL3 to collect your Fura- red. You can use FACS Assistant to get the ratio and export the data file, then look at using a graph making program like cricket graph (plots look pretty cool you can smooth them if they are too spiky). When you are loading you can add both dyes at the same time so it doesn't take any longer. We ppublished some work a while back in J.Exp Med 188 pp2057-65, I think we put our protocol in there somewhere. Simon Monard ADARC NYC Thank you for you comments. Beth Whalen U.B.C. Pulmonary Research Lab. St. Paul's Hospital Vancouver, B.C.
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