I added 0.5 microgram LPS to DMEM+FCS+PS and then incubated 24h. After that, I transferred the LPS-stimulated DMEM to RAW264.7 macrophages and saw a stimulation in cytokine production.
However, when repeating the experiment I used the same DMEM+LPS that I stored 7 days at -20; the second time there was no stimulation.
Could it be that the LPS after freezing is captivated by the tube and therefore no LPS was transferred to my RAW-macrophages the second time? Or is there another explanation why the LPS was inactive the second time?
hi milena
I think that freezing affect the physical form of LPS, some chemical can crystallize under such conditions that may interfere with reaction with TLR of macrophage....
Dear Milena,
LPS can not be freeze and thaw several times. The LPS stock in HBSS medium can be used once if freezed but not repeatedly. I think the DMEM also may not be suitable medium for stock preparation and freezing. I suggest you to use fresh LPS every time for treatment.
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