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HaCaT cell line - not responding to inflammatory stimuli such as LPS/TNF-alpha


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#1 Immunology_AlkM2014

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Posted 02 April 2014 - 08:23 AM

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?

 



#2 Curtis

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Posted 02 April 2014 - 09:09 AM

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



#3 Immunology_AlkM2014

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Posted 02 April 2014 - 09:17 AM

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



#4 bob1

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Posted 02 April 2014 - 11:39 AM

Have you tried fresh batches of LPS and TNFa?



#5 Immunology_AlkM2014

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Posted 03 April 2014 - 07:59 AM

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






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