I am studying about stimulating Hacat by LPS that is 0, 20, 40 ug/ml (in serum free media). After LPS treatment, it had 30hrs incubation time. ELISA was tested by IL-6 and TNF-a. However, it never changed than 0 ug/ml treat group.
So, I think a few possible problems.
1. Hacat (I got this cell line from colleague's lab)
2. LPS (E.coli species): sigma O127:B8
Who know about this problems?
If I want to get new Hacat cell, how can I usually do that?
Thank you for reading.
There may be few possible issues to consider:
- your LPS concentration seems fine, thats ok
- HaCaT are responsive to LPS, thats ok too
- you probably should stimulate with LPS for 12-24 hours (i cannot rule out how long the LPS stimulation was applied... -30 hours? Then you removed the supernatants for ELISAs?)
- a total deprivation of FCS during the stimulation (if 30 h) may "suppress" the cells in their functions to produce cytokines
- after LPS stimulation, the cells may require further incubation period of few hours prior sampling for ELISAs...
- TNFa is expected to have low signals in HaCaT by your approach, however, IL-6 should give a signal. IL-8 may give an even stronger signal than IL-6...
Optimization of treatment time could help. The time you used is too long.
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