mitochonrial activity, how to assay ?

kalifa ! juschus at novsrv1.pio1.uni-heidelberg.de
Tue Dec 19 02:49:02 EST 1995


Ahoy,

we are using the boehringer XTT cellproliferation assay which,
in principal is similar to the MTT assay. that is that
the activity of the mitochondrial GAPDH gene activity is 
used as an indicator for cellnumber.
our set up is as follows:

cultivate cells
remove FCS, add growth factor (FGF2)
wait 24 hours
add XTT assay mix to wells,
wait a few hours
carry out elisa.

So a rise in absorption would me a rise in proliferation.
However, under the miscroscope, we observe that some cells
become braun, then dark braun, black and finally lyse
with there cell body full of presumably the product
of the XTT assay. This predominantly in FGF2 treated cells, 
to a much lesser extend in control cells. 

We know (by counting life cells) that FGF added to the culture
doubles the number of cells after a 72 h incubation.
We now also know (from the XTT assay) that FGF apparently has
an effect on the mitochondrial activity.

questions: 
1. anybody out there who has used MTT or XTT assays
who has observed something like we did ?
2. we wanted to use the XTT assay as a simple 
means to determine proliferation. Are there alternatives that 
may work better ?

---> 3. How can we investigate the (interesting) effects of FGF on 
the mitochondrial activity ?

clemens
could you e-mail me directly at the above mentioned e-mail number ?




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