I am planning to do some experiments in which I need to determine the levels of neuroinflammatory markers such as IL-6, TNF-alpha and IL-1beta in mice brain.
Dear Mr. Gaurav,
You can use BV2 microglial cells which are derived from mouse. You can stimulate the cells with different toxicants such as lippopolysaccharide (LPS). Then you can collect the culture medium and then do ELISA. One more thing you can do. After direct treatment of the toxicant in the brain you can sacrifice the animal and collect the brain. Then make brain homogenates and do western blot and/or ELISA.
Hope this will help you.
Dear Gaurav,
First, I think you should perform the analysis in specific areas of your interest, otherwise you risk to not find any interesting and explainable results.
There are different inflammatory marckers that can be tested in several ways. There are, for example, ready-to-use ELISA kits that simultaneously test a series of cytokines, or other kits specific for a cytokine (TNF-α, IL-1β). Or, you can perform a quick and easy qualitative PCR analysis, since these molecules are produced on demand. I would add the immunohistochemical analysis later on, to support the results collected with other tecniques, that, in my opinion, could be enough.
I hope I have been helpful.
Dear All, Thank you for your reply. I am interested in a quick quantitative method to determine neuroinflammation in brain regions like Amygdala, PFC and hippocampus. I am interested in RT-PCR and ELISA kits. I am less interested in immuno-histochemistry at this stage. Can anyone of you give me details of RT-PCR? protocol? Thank you Moniruzzaman , Killol and Maria.
@ Maria- are you doing RT-PCR in your lab?
Dear Gaurav,
I'm actually performing RT-PCR, and I think that if you got primers, mixes and homogenate tissues, you are ready to work; In a couple of weeks you can already have some results. Maybe amygdala takes longer, because it is a very little area, with not too much RNA; so it request lyophilization before working.
Anyway I could help you.
Wishes
Maria Rosanna
Hi Gaurav,
A good way to measure several cytokines at the same time from brains is to use CBA (cytokine bead array), you can read the plate with a FACS.
It is a fast and efficient method, and you can save tissue material because you measure all the cytokines (present in the kit) at the same time in the same well.
You can purchase such kits from BD Biosciences.
Sighild
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