I'm trying to induce caspase activation in primary neurons with staurosporin treatment. Can someone help with what concentration I should start with and duration of treatment to see an easily detectable cleaved caspase 3?
Also, what should be the maximum dmso concentration that will not affect my experiment and control. I'll appreciate any help.
Thanks
6h treatment with 1 micromolar staurosporine should be enough to see cleaved caspase-3 by WB (in vitro - in culture). I always use less than 0.5% final concentration of DMSO (so less than 5 microliters in 1 ml).
Staurosporin is not indicate for in vivo studies, use it for just for in vitro experiment.
6h treatment with 1 micromolar staurosporine should be enough to see cleaved caspase-3 by WB (in vitro - in culture). I always use less than 0.5% final concentration of DMSO (so less than 5 microliters in 1 ml).
I wanted to give the exact same answer as Saska did, 6h with 1 µM stau has been right for most cells I studied so far. And 0.5% DMSO should be ok as well.
I used 1 microMolar for 24h on Caco-2 differentiated monolayers (That are much more resistant than primary neurones) with noticeable results. You can check the images here (Figure 3E and 5E): http://www.mdpi.com/2072-6651/6/1/211/pdf
I hope it helps you.
My collegue Abdelhaq Rami and his coworkers used 300 nM (0.3 µM) Staurosporin for 1, 3, 6 and 12 hours in primary hippocampal neurons and HT22 cells (Spahn et al., 2008; Neurochem.Res). Be aware that Staurosporin is a rather "dirty" drug that is doing much more than inhibiting PKC (see papers from Sir Philipp Cohens group in the Biochemical Journal).
I think you're referring to in vitro culture. In this case, I would use 50 nM. You should Perform with this concentration a kinetic curve. Micromolar concentrations can induce cell death. You have to have carefully.
I would also suggest to start your experiments with rather low concentration at a nonomolar range. You should try different concentrations, e.g. starting with 50 nm ,250 nm, 500 nm and not exceeding 1 µm and different incubation times up to 12 or 18 h
Try a range of concentrations from 0.25 micro molar to 1.0 micro M as indicated in previous studies.
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