For the past few weeks we've been using three different cell types on the Fura 2 calcium experiment including iPSC's, RenCells, and human skeletal muscle cells. We loaded our cells with 2.5 uM of Fura 2 in HBSS + 1% BSA. We incubated the cells in both 37 degrees or at RT for about 30 minutes and hydrolyzed for about 20 minutes in HBSS. Using the Olympus microscope and MetaFluor software we were able to see the cells with the fluorescent microscope indicating the loading was successful, but saw no response when exposed to the drugs we used. As a positive control test we used 5mM glutamate or 20uM ionomycin and also saw no response. I appreciate if anyone could give any insight on potential issues or solutions we can take to successfully run this experiment.
Did you load the cells using the acetoxymethylester form of Fura-2? Your comments suggest you did. Use fresh DMSO to solubilize the Fura-2 AM. Which excitation filter set are you using? Which emission filter? Also ensure your cells are in glass and not plastic chambers. Plastic absorbs UV wavelengths more so than glass.
If you can't get Fura-2 to work, switch, at least temporarily, to using Fluo-3 or Fluo-4. They are easier to work with.
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