Should I perform MTT test to analyse the effect of increasing concentrations of Lipopolysaccharide (LPS), before induction of inflammation with one specific concentration? I can choose a treatment plan and concentration of LPS reported in some related articles. I'm reading many papers related to cell culture models of inflammation. Concentrations are from 25ng/ml up to 100ng/ml, but it depends from the treatment timealso (from 4h up to 24h). Do you have any comments on that?
How can I choose the best concentration of LPS to treate cell culture (J744A.1 macrophages) and the best duration of treatment with LPS, to get good induction of inflammation? Do you have any suggestions?
From my experience with RAW macrophages (mouse macrophage cell line similar to J774A) the optimal concentration of LPS for a strong inflammatory response induction is between 500 and 1000 ng/ml. You will find in literature concentration much lower than these (100 ng/ml) but normally they are used in primary cell models (more sensitive to LPS stimulation).
A simple, rapid and cheaper way to access if you are effectively inducing an inflammatory response is by measuring NO production. In my lab by routine we use 1000ng/ml during 24h and then check NO production by Griess reaction.
Regarding the exposure time it will depend on the output that you want to analyze:
-For MAPK and nfkb activation: normally 15 to 30 minutes of exposure
- For effects on the gene transcription (citokines, chemokines, etc…) 6 hours
- For NO production, COX2 and iNOS protein expression: 24h
Because of different stimulation and cytokine expression patterns in every cell type, it's always important to make some time kinetics within your experimental setup before starting with your real experiments.
Just take different concentrations from, e.g., 10 ng/µl up to 500 ng/µl or an other dilution series and repeat this also with different numbers of J774A.1 (e.g. 5 x 10^4 up to 1 x 10^6 cells) and check for IL-6 or TNF-a doing sandwich ELISA. With this, you can easily figure out the best concentrations and cell numbers for your setup.
Dear Olav,
Firstly, it could be made a growth curve of these cells. These curve let you know when the cells are in exponential phase where the cells are usually more sensitive to your treatment.
Once determined the growth curve, you can choose the inflammatory marker (as Dogan said IL-6 or TNF-a would be excellent cytokines), treat the cells with different concentrations of LPS (Zhu et al., 318, 2012, Exp. Cell Res. treated myofibroblasts of colon with concentrations up to 1ug/mL of LPS), and make and ELISA to investigate the effect. According to these results you can select the concentration that better results gives you.
MTT will give you no information about the inflammatory effect but you will see the possible cytotoxic effect of LPS on the cells.
In attachment you can find an article the monocytes-derived macrophages were treated with LPS.
If you have any doubt, please ask me.
http://www.ncbi.nlm.nih.gov/pubmed/22683859
From my experience with RAW macrophages (mouse macrophage cell line similar to J774A) the optimal concentration of LPS for a strong inflammatory response induction is between 500 and 1000 ng/ml. You will find in literature concentration much lower than these (100 ng/ml) but normally they are used in primary cell models (more sensitive to LPS stimulation).
A simple, rapid and cheaper way to access if you are effectively inducing an inflammatory response is by measuring NO production. In my lab by routine we use 1000ng/ml during 24h and then check NO production by Griess reaction.
Regarding the exposure time it will depend on the output that you want to analyze:
-For MAPK and nfkb activation: normally 15 to 30 minutes of exposure
- For effects on the gene transcription (citokines, chemokines, etc…) 6 hours
- For NO production, COX2 and iNOS protein expression: 24h
I do agree with Dogan answer. You should perform kinetics and LPS dose/effect to be sure that your stimulation/incubation conditions are optimal to detect your cell response. For instance if your aim is to measure TNFa, you should collect your culture superntants at different time points (0, 2, 6, 12, 24 hr post stimulation) with varying concentrations of LPS ranging from 10 ng/mL to 1 µg/mL. You can start with 1.10e5 J774 per well in a P96 culture plate. Hope it helps.
Cheers !
Dear Colleagues,
Thanks a lot to all of you! I will keep you up to date with the results!
yes people I am working on LPS as a part of my Phd thesis too. I appreciate any help on dosage
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