HI everyone,
I've been doing some signalling experiments with murine bone marrow derived macrophages with LPS and I was hoping to repeat them using IL-4 as a stimulant. I normally use 100ng/ml for 0, 10, 30, 60, 90, 120 and 180 minutes then lyse cells in RIPA buffer and prepare protein lysate samples for Western Blot.
I'm hoping to basically repeat exactly the same with Il-4 however I have very little experience in IL-4 stimulation and was hoping you would be able to advise on appropriate concentrations? I was thinking 5ng/ml of IL-4 however I've seen some papers use up to 20ng/ml. I'm looking for a concentration that will give me a signal that is not high enough to activate pathways other then the IL-4 pathway.
Many thanks for your help!
I have done IL-4 titration stimulation on BMDM recently; I found 1ng/ml is the lowest concertration to activate downstream gene expression, but when I used 10ng/ml, the stimulation didnot increase the gene expression anymore. Therefore, I think 5ng/ml is a proper concentration for your experiment.
The lowest I would use is 5ng/ml and highest if you want to reach plateau activity 20-40ng/ml The kinetics will be prolonged as the dose will decrease, meaning higher concentration of IL4 will give you faster kinetics.
For B cell preliferation experiment, I usually use 20ng/ml to stimulate B cells. it works very well.
Good luck,
Xuefang
The commonest concentration for IL-4 stimulation in BMDM is 20 ng/ml but that is in the context of M2 activation, which occurs over several days.
Thanks everyone for all your helpful thoughts. I think I might just do a titration at first. I'm only looking at very early signalling events (so 5, 10 mins) at least for this experiment though I am hoping to look at M2 IL-4 phenotype of the cells cytokine wise later on. I didn't realise you needed to have IL-4 over several days I thought it would be the same as LPS and I could do a 12 - 24 hour activation to stain for cytokines via flow cytometry. Thanks for mentioning it Fiona or I would have got all the way down the line and not realised! =D
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