We tested LPS concentrations of 0.5, 1, 5, and 10 μg/mL to stimulate the cells for 12 or 24 hours, but qPCR detection showed no significant changes in inflammatory factors.
We hope researchers with experience can provide suggestions. Thank you in advance.
Hello morning,
In my opinion, i recommend optimizing the conditions by testing:
- Longer times (24-48h).
- Higher concentrations of
LPS (up to 100 ug/mL).
- Pre-activate the cells with cytokines.
- Confirm the expression of
TLR4.
Hi Cheng,
in addition to what Adriano Dala Cassanje said I would also advice that you determine if you see any changes at the protein level by ELISA (what I would expect). In my experience this is often easier than doing qPCR.
Regarding the qPCR - do you have a good positive control and a good negative control showing that the qPCR itself is working properly? Especially with really high CT-values you should consider problems with qPCR itself.
I would be careful regarding the really high LPS concentration - they might induce apotosis in NCM-460 cells (please check this publication: (PDF) Gold nanoparticles alleviates the lipopolysaccharide-induced intestinal epithelial barrier dysfunction - there is a full-text link on Research gate).
Cheng Cheng , This is very strange. I wonder which inflammatory factors you chose to verify?
Because we have verified the dose of LPS to induce cell inflammation, which is generally around 1 μg/mL. After 24 hours of stimulation, the mRNA expression of inflammatory factors can be detected significantly increased.
And we found that this article mentioned that NCM-460 cells were treated with 1μg/mL LPS for 24 hours to construct an inflammatory model. This study used dexmedetomidine as a positive anti-inflammatory drug to detect the protein levels of TNF-α and IL-8 (ELISA), as well as the expression levels of cyclin D1, cleaved Caspase 3 and key proteins in the PI3K/AKT signaling pathway (WB).
10.3969/j.issn.1007-7146.2024.04.010
Check your validation indicators and change the validation method. Dörthe Andrea Kesper may also suggested this.
It is not recommended to increase your treatment concentration, and it is difficult to control the treatment time. On this point, I have a different opinion from Adriano Dala Cassanje .
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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