Fura Red and Fluo 3
Martin Poot (MARTIN@probes.mhs.compuserve.com)
03 Jun 95 11:35:07 EDT
We have limited experience with calcium measurements. In ouur hand Fura Red
and Fluo 3 can be loaded simultaneously into cells using 10 to 20 uM Fura
Red and 1 uM Fluo 3; this gives intensities on the same scale. The quantum
yield of Fura red is very low (near 0.01), so it may be hard to detect the
signal unless loading concentrations are relatively high. We prefer to load
at 4 degrees Centigrade for 15 to 20 minutes, then try to calibrate the
signal by the standard methods used for Fluo 3 and Fura 2 (varying
extracellular calcium and using ionophore). May be you start with Fura Red
alone and see how much dye you need to obtain a reasonable signal. The try
the combination of dyes. This setup assumes co - localization of dyes and
consitent loading between different sets of cells. Since the intracellular
compartment will change with time, the ratio of green to red signal will
change. It would be good to assess dye loading concentrations, temperature,
and time in the microscope. Loading time should not be longer than one hour,
although cell types may vary. Inhibitors of anion transport like verapamil
and probenicid may influence loading. The problem with the proposed 90
minute experiment may be that there is still a fluorescent signal, but the
dye is no longer in the cytoplasm and hence, little or no changes in
cytosolic calcium are recorded.
Good luck,
martin@probes.mhs.compuserve.com
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