I am looking at the NF-Kappab inhibition in fibrobalsts. In one of the references from the inhibitor data sheet i found that the cells were incubated with the BAY-11-7085 inhibitor at a concentration of 10 microMolar for half an hour prior to the incubation with the stimulant. Kindly advise if anyone has used this inhibitor for the irreversible inhibition of NF-Kappab and how was the inhibitor applied? What criterias have to be kept in mind for such an inhibition strategy ?
The inhibitor (BAY 11-7085) is dissolved in DMSO and a working concentration is 5-10 micromolar (not 10 mM). The inhibitor inhibits the activity efficiently in most cell types. Use equal volume of DMSO in control cells.
In my lab Bay was commonly used with a concentration of 10 µM disolved in DMSO. We directly applied it into the cell medium one hour before we used the stimuli. If you are not sure it is working properly (it may be past date or whatever, we kept it at -80ºC), you may also try parthenolide at the same final concentration, since it also inhibits NF-kappaB.
It must be a typo for 10mM. I suppose it should be 10uM.
Sometimes Copy/Paste in Microsoft Word may change the Greek symbol u(micro) to m.
well thats what happened, i changed it now. Anywaysw thanks for the suggesions. Carlos I also think thats its better to store it at -80 degrees as the solution can be stored for 6 months approximately. In case the solution is thoughed it can be stored at 4 degrees for 2 weeks. I decided to use it for 1 hour before the stimulation of the cells and then look at the activation/ inactivation of the nfkappab pathway. I am taking different concentrations of 300, 30, 3, 0.3, 0.03, 0.003,and 0.003 micro molar. I think i can decide upon a best concentration to be used post such an experiment.
we used BAY-11-7085 in our lab before and found that BAY-11-7085 does not work in any cell line or for any cell type. I would suggest to try different NFkB inhibitors side by side and select the most efficient one.
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