I have a question about coupound treat on Raw cell.
My Plan is treating LPS 6hours and then compounds for 24hours on CTL-3 well, LPS-3well, Compounds-3well, LPS+Compounds-3well.
I need your help to know how to treat LPS. I mean i have to treat it for first 6hours of total 30hours treatment or Last 6hours of total 30hours?
I think the role of LPS is like inducing pro-inflammatory cytokines. But, I think i have to treat for first 6hours and then change to normal dmem for next 24hours to compare LPS+Compound groups. I also think that it will show different effect between both treatment becasue LPS 6hours + dmem 24hours and dmem24 hours + LPS have different cytokines levels.
I really want your thouhgts
Hello Sungug Joo
Lipopolysaccharide (LPS) plays an important role in inducing the inflammatory response. Exposure to inflammatory stimulants, such as LPS, triggers transcription of proinflammatory cytokine genes like TNF-α, IL-1β, and IL-6.
You could consider five groups for your experiment.
Group 1
You may treat RAW264.7 cells with LPS for the first 6 hrs. to mimic the chronic inflammatory environment and then treat the cells with the compound for the remaining 24 hrs. If your compound is anti-inflammatory, it will significantly inhibit the levels of proinflammatory cytokines that were induced by LPS.
Group2
Also, you may try pre-treating the cells with various concentrations of the compound prior to LPS challenge. You may find that by doing so, it may notably attenuate the enhancement of the proinflammatory cytokine secretions.
Group3
You may also have another group wherein you treat your cells with LPS for first 6 hrs. and then change to normal DMEM without the compound for next 24hours.
Group 4
You could add one more group wherein you treat your cells with the compound only without LPS stimulation.
Group 5
You may also consider another group wherein you leave your cells in DMEM without any treatment, neither LPS stimulation nor treatment with the compound.
Good Luck!
in my opinion, i would also include a time course of your compound in the presence of the LPS challenge and with LPS alone. you may find that in RAW cells, the peak response to LPS is sooner than 6 hours. my lab does an ex vivo assay in which we add LPS to extracted whole rat blood and incubate for 4 hours to observe a maximal increase in plasma TNFa.
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