I have stimulated primary human monocytes and monocyte-derived macrophages with 100 ng/ml E.coli-derived LPS for 24 hrs (monocytes) and 24/48/72 hrs (macrophages). Total MMP-9 ELISA on cell-free supernatants has shown no increase in MMP-9 for any donors, actually many have shown a reduction/MMP-9 below unstimulated control levels. It seems well-known that LPS drives MMP-9 production from these cell types so I don't know why I'm not getting the same results?
*I have isolated primary human monocytes from healthy volunteer blood by Ficoll/density centrifugation and plastic adherence. Macrophages were generated by 5-7 day incubation in 10 ng/ml rhGM-CSF in 10%FCS/RPMI1640.
The LPS is fresh, has been giving "typical" LPS responses in others lab work (not looking at MMPs), and I've tried aliquots from multiple different stocks. I didn't reconstitute it myself, but it was done by other competent lab users.
Hi Sarah, the main problem in looking for LPS specific response is that everything has some LPS on it or in it. Water, buffers, salts, tubing, tubes, gloves, you name it, all have some LPS.
Unless it has been certified as pyrogen free, serum is about the only fluid that does not have LPS (if it has been properly handled).
1. make sure that your media are not contaminated with LPS as outlined above.
2. 100 ng/ml LPS may not be enough - try 1 ug/ml and 10 ug/ml
3. make sure that you have serum (10%) in your culture medium
3. Have you done a time course experiment to make sure you are looking at the optimal time point..
I do not entirely agree with Caroline in using higher concentrations of LPS to elicit a response in isolated monocytes/macrophages.
PBMCs are very sensitive to any LPS in the environment. Their response is usually either all or nothing.
If the monocytes/macrophages have been exposed to LPS prior to isolation, they might be spent and are not be able to respond to any more LPS.
A simple way to assess whether the cells are 'spent' is to measure another robustly induced cytokine such as CXCL8. At the time points you have analysed this should still be present at high levels.
The low level of FCS could effectively be 'starving' your cells. Your cells could be quiescent or even dead. Have you conducted a viability assay?
I agree with Caroline that the time point may also be a potential reason so I would suggest a time course. Another option is to measure gene expression - is LPS in your system actively inducing MMP-9 production or release?
Good luck!
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