Calcium detection Questions....

James J. Campanella jjc4 at po.CWRU.Edu
Thu Mar 13 11:21:01 EST 1997


Hi,
 I'm putting together a protocol for a cell biology laboratory class
and the texts that I have do not answer some of the questions that have
come up. The lab simply involves examining animal cells that have been
treated, and not, with an ionophore induce calcium flux. The students will
then stain the cells with Fura-2 and examine induced and uninduced cells
under the fluorescent microcope. Here are some of the questions that have
arisen in my mind:

1) Can you treat animal cells with a calcium ionophore like calcimycin, 
fix them, and then demonstrate the presence of the calcium under a 
fluorescent microscope? The real question is the one of fixation...

2) What calcimycin stock concentration would one use? What is the usual 
length of cell incubation?

3) We will use Fura-2 to visualize the calcium ions. Is there a better
calcium ion probe than this?

4) Is it better to not fix the cells? If unfixed how long before the calcium
 ions are pumped out?

5) Can anyone suggest a decent background text or manual on fluorescent
histochemistry?

Thanks for the help,

Jim Campanella
Lehigh University
Department of Biological Sciences
Bethlehem, PA 18015
USA
-- 
**The Past tempts us, the present confuses us, and the future
  frightens us . . . and our lives slip away, moment by moment,
  lost in that vast terrible in-between** 
                                  -The late Centauri Emperor



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