We are currently having some problems with DAF2 or DAF-FM fluorescence. We use a 5µM concentration and are not able to see any effect of a stimulation on DAF fluoresnce. When looking cells under the microscope, the fluorescence appears weak as if some of the probe was leaking after their washing. Is it possible to perform the quantification in culture medium (seems to be the same with PBS or medium), is it a problem to havec 5% DMSO in the well ? We use L-arginine without any difference.

Currently 30min incubation (5µM)/ washed with PBS or medium/stimulation with bradikinin/acetylcholine/caclium ionophore/ATP... No reaction.

Thanks