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WE have been using a Coulter Elite and Indo-1 to monitor cytoplasmic Ca in
human T cells. Until now we have been using Ca containing buffer so that the
bulk of the calcium measured is the flux into the cell across the plasma
membrane. We would like to be able to measure also just the release from
intracellular store to the cytoplasm. We have tried doing this by measuring
the flux in cells suspended in Ca-free (either Chelex-treated on EGTA-containing
), but we appear to get no measurable flux. It may be that the system is not
sensitive enough because of the UV laser, which is very difficult to focus
as tightly as the other lasers and so our quantam yield is lower.
Has anyone tried to do such an experiment? Are there any advantages in
sensitivity in using, say, fluo-3? Any tips would be appreciated.
Thanks
Steve Young
dept of Rheumatology
Univ Birmingham, UK
Email to s.p.young@bham.ac.uk Voice: 021-414-6480
***END-OF-MESSAGE***
Steve,
We have done as you outlined using EGTA to chelate external calcium
in the media. We have seen good results with this system using indo-1.
But we have seen flatline responses also. How large of a deflection
do you see on your system (with external Ca++) using Ionophore, then
followed with EGTA? If your dynamic range for this system control is
compressed, you might have trouble seeing an internal mobilization. It
is usually smaller and of different kinetics than external influx.
Peter Lopez
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Peter A. Lopez (617)632-3179 (voice)
Dana Farber Cancer Institute (617)632-3139 (voice)
Core Flow Cytometry Facility NO FAX!
44 Binney St. Room J-312
Boston,MA 02115 PLOPEZ@sorter.dfci.harvard.edu
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