I am going to do some neuron and microglia coculture and i need positive control for neuron activation. I tried to test the P-ERK by western blot after AngII treatment. However, it seems no change. Actually the level of P-ERK in naive group is also high. And i also want to try Ca+ measurement? Is that possible to do so?
did you starve the cells before stimulation with AngII? There are lots of things in serum that would increase basal P-ERK, and that what you cannot see any additional increase with further stimulation.
Ca+ measurements can be done easily my microscopy, you would need to load the cells with Fura-2, Flou-4 or some other calcium indicator (see Molecular Probes website for details)
Hope this helps
It depends of the cell type. Check in the literature for neuron and microglia. I usually starve cell lines and tough primary cells for at least 18h (overnight), stimulation is also done without serum. Some cells are bit more fussy, so they need to have some serum (2% or charcoal striped).
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