Hello Erjola Rapushi

Stimulation of PMA-differentiated THP-1 cells with 0.1 ng/ml LPS may be insufficient. Cytokine release by PMA-differentiated THP-1 cells is dose dependent. Try using LPS concentrations at 1, 10, 100 and 1000ng/ml for 24 hours.

For more information you may want to refer to the paper attached below. See Fig 1E.

In this paper, PMA-differentiated THP-1 cells were treated with different concentrations of LPS ( 1, 10,100 and 1000ng/ml) for 24 hours.

https://journals.sagepub.com/doi/10.1177/17534259221100170?icid=int.sj-full-text.similar-articles.7

Also, you may be interested in the article attached below. See Fig 3. In this paper, in differentiated THP-1 cells, stimulation with 1 and 2 µg/ml LPS at different time points (6, 12, 24, 48 hours) resulted in an increase of TNF-α levels compared with control.

https://www.spandidos-publications.com/10.3892/mmr.2018.8542#:~:text=In%20differentiated%20THP%2D1%20cells,cells%20following%20LPS%20stimulation%20(Fig.

Best.

Malcolm Nobre my mistake, i meant 0.1μg/ml LPS. That was the concentration of LPS used for stimulation and didn’t get detection of TNFa. I am using fresh LPS every time.

Once LPS is thawed, it should not be frozen again.

Freeze-thaw destroy it.