HaCaT cell line - not responding to inflammatory stimuli such as LPS/TNF-alpha - (Apr/02/2014 )

Hi

We have been using HaCaT cell line in our lab for long to check cytokine secretion (IL-6, IFN-y etc) by LPS/TNF-alpha secretion. 

However now the cell line is not responding to LPS/TNF-alpha. There is no increase in secreted levels of these cytokines after stimulation. However, the cell line is still sensitive to anti-proliferative drugs...

What can be the possible reasons and how to solve this?

how did you do this? could this be because of low concentration?

We plate cells for 24 h till reaching confluence, sera starve the cells fr 24 h and treat cells with LPS (100 ng/ml, 1 ug/ml) or TNF-alpha (10 ng/ml, 50 ng/ml).

At these concentrations, we used to get good increase in cytokine secretion earlier. 

Could this resistance be due to increase in passage number??? not sure

Have you tried fresh batches of LPS and TNFa?

Not yet....But the same batch is showing good expression of cytokines in other cell lines such as RAW 264.7 and THP-1 ...:(