I'm going to stimulate the Human machrophages and monoctes with E.Coli LPS, I know 1µg/ml to 10µg/ml LPS could be effective, but I'm looking for the optimal concentration.
It depends on the conditions of stimulation and what you mean by 'optimal'. A key factor is the presence of human serum as a source of LPS-binding protein. For monocytes, it may be that they are less responsive due to the expression of TLR4.
In the presence of human serum (10% v/v) I would expect 10ng/ml to give a response (TNF-alpha production) above background in monocytes and macrophages. In differentiated cells, we see strong responses down to 0.2ng/ml. I would advise against using high levels of LPS as there can be off-target effects e.g. cell death and it is also non-patho-physiological.
The only way to be sure though is to titrate the LPS in your system, with your cells, with your density of cell seeding and with your 'read out'. All these factors will affect the precise concentration required to give you the response you need.
I agree with Andrew. The cell's conditions may be tricky also. The availability of healthy cells are important for you to get reproducible results.
1-10 ng/mL of LPS is more than enough to induce changes, but it depends on what your read out is and what you're assaying for. The only way to truly find the "optimal" concentration is to do a titration and use multiple concentrations of LPS.
I agree with Dionna Williams on doing a titration experiment. lalitha kabilan
1-10 ng/mL of LPS should be enough on both cell. Some considerations about the dose include if you are going to work with serum starvation or not, cell density and the time of stimulation that you are using (design of the experiment). For instance if you are going to use pretreatment with drugs or co-treatment is better use lower conctr. of LPS due increased possibility of toxicity. you may need to do always MTS or MTT and depending of the target you may do titration. For THP-1 the concentration may variate as well if they are stimulated or not with PMA.
At 1µg/ml you will clearly induce a change...and kill your cells!
200ng/ml should be enough to activate your Mph in early time points (6-12h) for human monocytes- derived Mph. A titration is clearly the best option since it will depend on what you are following (activation marker, cytokines secretion...)
gook luck
Hi, second year undergrad here in need of help, why will cells die at a high dose of LPS (grew cells, PMA to differentiate, and LPS to stimulate). I used multiple concentrations of LPS to see the variations and the highest level didn't produce any TNF alpha. Searched for ages but think I'm on the wrong track altogether. Any help much appreciated as deadline is looming.
I know this is long past, but in the interests of information 50-100 ng/mL should be more than enough presuming these are primary cells and not a cell line. It's physiologically relevant and primary macrophages are much more sensitive than immortalized cells. For eg, in mouse macrophage line RAW 264.7, we typically use 1 ug/mL to get the same response as from BMM (0.1 ug/mL)
50 ng/mL works perfect in HAP cells and also normally does in monocytes/macrophages.
Hi all,
I need to treat primary cortical neurons with LPS for short time (range of 1-2h); which concentration you suggest to avoid the neurons death?
Thanks
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Usually, 10ng/ml LPS is enough for stimulation. And this is also our conditon for murine macrophage stimulation. Good luck!
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