I am trying to treat the 21 days differentiated Caco2 monolayer with LPS at the concentration of 5µg/ml.
During the differentiate stage (21 days), I feed the cells with DMEM 10%FBS, 1% NEAA, 1% Glutamax, 2.5% HEPES, 4.5g/L D-Glucose and 110mg/L sodium pyruvate.
After that, I use the HBSS media with 2.5% HEPES for LPS treating.
I add the LPS with HBSS media to the apical side of the trans well at the total of 450µl, and 750µl HBSS media alone in the basolateral side.
After 24hrs, the LPS did not show any effect on the TEER of the monolayer, but the cells is dying at this time due the serum free media HBSS.
Normally LPS take long time to have some effect (More than 48hrs I think) but I cannot change the media in the middle of experiment.
Should I wait more for the LPS to take effect or I should change it back to the growth media (DMEM) ? Because if the monolayer is damaged, it's definitely affect the TEER value.
You could try treating with serum-free LPS media for a few hours, then replacing with fresh LPS-free media.
A greater concern is that Caco-2 cells have been reported to be unresponsive to LPS treatment (lack of TLR4 expression):
Inflammatory parameters in Caco-2 cells: Effect of stimuli nature, concentration, combination and cell differentiation.
http://www.sciencedirect.com/science/article/pii/S0887233310000858
Responsiveness of intestinal epithelial cell lines to lipopolysaccharide is correlated with Toll-like receptor 4 but not Toll-like receptor 2 or CD14 expression.
http://link.springer.com/article/10.1007%2Fs00384-002-0415-6
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