Q: Measuring Ca release in transfected cells.

Wolfgang Schechinger wgschech at med.uni-tuebingen.de
Sun Aug 16 06:29:31 EST 1998


Hi all, 

I'd like to make a "quick and dirty" test for changes in sensitivity 
on growth factor stimulation.

Quick and dirty means that I want to load the cells with FURA2, 
put them into a fluorimeter cuvette, add growth factor and see what 
happens to calcium efflux. (At the moment, I don't have the equipment 
for higher sophsticated experiments like a confocal microscope etc.)

The crucial point is the my cells actually are adherent 3T3 mouse 
fibroblasts and need to be transfected.

My plan was:

1) transfect cells
2) grow cells for another 24h
3) detach cells by a short trypsination and mechanical force, count 
cell number 
4) keep cells in suspension for 4 hours (agitate in the 
incubator, either constantly or by swirling every five minutes)
5) load aliquot with FURA2, place into cuvette, stimulate 

All I want to see is if the transfected plasmid (and 
expressed protein, of course) affects the ability of the cell to 
produce calcium efflux (duration / mean intensity / recovery time) or 
not.

Will the 4h agitation be enough time for the cells to restore 
functianlity in their receptor system (for PDGF), since I 
assume that trypsin happily will chew the receptors into pieces?

All input is welcome!

Wolfgang
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usual disclaimers apply * This message is RNAse free - please don't touch!
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Wolfgang Schechinger         
University of Tuebingen, Germany
email: wgschech at med.uni-tuebingen.de * wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
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