I want to stimulate a Human endothelial cell line (HUVEC) by LPS and test my drugs of interest to quantify the anti-inflammatory effect, I used the DMEM medium and 10% FBS for HUVECs, at first I test the viability of cells by MTT assay, for this I stimulated with 1µg/ml, 5 µg/ml and 10 µg/ml of LPS. I just use the 5 µg/ml and 10 µg/ml concentration to see if LPS has some effect on endothelial cells but there was no difference with the control group. Where is the problem? I bought the LPS form Sigma. Does the HUVEC stimulation need a special protocol?
The medium you use is not specific for HUVEC... What do you want to measure? cell death? you could first look at endothelial activation by e.g.measuring ICAM-1 expression. This should be increased following exposure to 1 ug/ml LPS for 24 hrs. you can email me at [email protected] for more details.
cheers,
Frank
Dear Frank and Thomas, my thesis is the anti-inflammatory effect of antidepressant, I know I should measure the ICAM and VCAM expression for endothelial cell activation, but in protein level by flow or ELIZA is a little expensive for me, I measure them in mRNA level, but I couldn’t get a desirable result, and I think maybe LPS cannot work and may be this protocol for endothelial cell stimulation is not good,
I think if the LPS can activate NF-ƘB pathway, at least, it should be effect on cell viability in 10µg/ml, but no difference with control was observed…..
Please tell me how can I stimulate my HUVEC by LPS?
Thanks for article, it was useful, the antibody is expensive, but may be the evaluation of mRNA level of ICAM and VCAM is not enough and I should try to buy their antibody..
can anyone tell me how LPS act on endothelial cells as we know endothelial cells lack CD14 (mCD14)???
@Laleh Rafiee, I am facing the same issue, how did you deal with it?