From: Eric Martz (emartz@microbio.umass.edu)
Date: Tue Aug 02 1994 - 18:40:57 EST
Can you guys at Molecular Probes (or anyone else) tell me: 1. What is the fluorescence intensity ratio for emission of calcium-saturated fluo-3 vs. fluorescein? 2. Is fluo-3 emission pH sensitive as for fluorescein? Knowing #1 would allow me to estimate, from the molecules of equivalent soluble fluorescein (MESF)/cell, the absolute concentration of fluo-3 in the cell (assuming uniform distribution). In our case, we're exciting with a 488 nm laser, and detecting in a 530 +/- 30 nm filter. But I just want a rough idea. Assuming the emission ratio is 1, loading lymphocytes with fluo-3 at 0.04 micromolar for 10 min, I calculate that the intracellular concentration is about 0.2 micromolar. This gives us a signal to noise ratio of 5 for basal cell fluorescence (no stimulus nor ionophore) where noise is autofluorescence, i.e. quite workable. MESF is obtained by reference to commercially available standard beads (Flow Cytometry Standards Corp.). Fluo-3 signal goes up about 10-fold with ionophore. /*- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Eric Martz, Professor of Immunology emartz@microbio.umass.edu Dept Microbiology Voice: 413-545-2325 FAX: 413-545-1578 Morrill IVN 203, Box 35720, Univ Massachusetts, Amherst MA 01003-5720 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -*/
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