Dear colleagues, I would like to test the quality of my primary antibodies by WB. I look for mouse TNF alpha and IL-4. Could you please help me how to stimulate mice by LPS and Concanavalin A in vivo to get high concentrations of these cytokines? I am interested in timing and dosage. Thank you in advance for your answers.
Hi Fabian, I have injected LPS (60ug) i.p. and later analyzed the mice after 12 hours to analyze invivo DC migration. Injecting LPS i.p. results in systemic activation and is also suitable for analyzing cytokines. I have not used conA for this purpose.
Hi,
For LPS-TNF-alpha, we use LPS 50 µg/kg (i.v.) and TNF measurement after 1 h. For Con A, we use 20 mg/kg. Never measured IL-4 in both the experiments.
Thank you for your answers, I searched for many papers and they differ a lot in the dosages and time. Also many of people use these mitogens in vitro not in vivo. I think there is no general protocol for this type of experiment.
Hello
Look at these papers may help you
Good Luck
http://iai.asm.org/content/68/3/1014.full.pdf
http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2567.1996.450512.x/pdf
I wouldn't rely on LPS for an IL-4 antibody... IL-4 is produced primarily by T cells responding to type-2 inflammation. Try an allergen... TNF, sure. Con-A will give your mice a nice hepatic injury, and also not IL-4.
General protocols for LPS injection? There are about a million. Most of them essentially specify LPS, a needle, some PBS, and that's it. TNF peak around 4-6 hours, I believe. But you'll see it at 20, 2, 12, as well. LPS is nasty stuff. Check the LD50 for the mouse strain before you inject; some are very sensitive, some not at all. If you want to get complicated, you can consider serotype, ultra-pure (TLR4 only) or standard (TLR2 and 4), but for what you're doing, methinks not necessary...
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