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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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