Hi, I am using LPS (Escherichia coli O111:B4) to induce HaCaT cells for inflammatory respone. I am using the concentrations of LPS about 100-1000 ng/mL. And stimulus time is 6-24 hours. However, my LPS is not inducing inflammation in HaCaT cells, but the same LPS did induce an inflammatory response in the Raw 264.7 cells！！！ Has anyone ever experienced this before? Is there another LPS used to induce HaCaT cells for inflammatory respone? I try to search references on line, but no reference can help me to solve this problem. Do you have any idea about this question?
Hey Yanting,
involving RAW macrophages as a positive control was a good idea. So your LPS is definitley working. You used LPS concentrations with 1µg/mL as the highest concentration. For macrophages this works, because, as a immune cell, they react much more effective. But I searched in literature a little bit and I found LPS concentrations for HaCaT cell ranging from at least 10 µg/mL to 25µg/mL. So perhaps your used concentrations might just be too low for HaCaTs (as a kerationcyte, non-immune cell) but good for RAWs (macrophage-like cell line, immune cell).
You can have a look at this paper for further ideas, if you like:
Anti-Inflammatory Properties of Low and High Doxycycline Doses: An In Vitro Study, 2015
Roberta Di Caprio, Serena Lembo, Luisa Di Costanzo, Anna Balato and Giuseppe Monfrecola
All the best and a happy new year,
Marc
Marc Herb Hi, Marc, thanks for your answer and advise. I have read this article before. I'm thinking about whether this high concentration of LPS causes HaCat cells death.I also read some literature in which the author used such a high concentration ( 10 µg/mL to 25µg/mL ) of LPS to stimulate HaCat cells and then studied the indicators related to apoptosis. So I wondered if there was another type of LPS (at lower concentrations) that could stimulate inflammation in HaCat cells. I will conduct relevant pre-experiments according to your suggestions. Thanks again for your kind answer.
Best wishes to you and happy new year
Yanting
Hi Yanting Wu
I am having the same problem with you when using LPS (Escherichia coli O111:B4) to induce inflammation on HaCaTs. Could you tell me whether your experiment worked after increasing LPS concentration?
Hi Quynh Nguyen
Because of the COVID-19 outbreak, I haven't done this project for the time being
Hi Yanting,
I performed anti-inflammatory studies with both RAW cells and HaCat cells. As Marc suggested we used to do with different LPS concentration. for Hacat cells i used 5ug/mL whereas for RAW cells i used 20 to 50ng. i got good results in ELISA with this concentration.
On additional note, you can also use LPS 055:B5 in case you are using cell lines other than RAW macrophages. Please update us whether you succeed after trying this. Good luck.
Dinesh Kumar
Thank you for sharing. I do not use this cell line for inflammatory studies at present. If I succeed next time, I will share my results with you.
Best wishes to you.
Hi Yanting Wu ,
I have tried increased LPS concentration to 20 ug/mL and treatment for 24h, the inducing effect was good on chemokine (TARC/CCL17) and cytokine (IL-8) markers in my RT-PCR experiment. HaCat cell viability decreased to 75%, and I did not observe cell morphology changes.
I will update more information if I can obtain western blot result. I think you can try this.
Hi, A basic biology advice would be to first check whether HaCat cells expresses the components responsible for triggering LPS/TLR4 signal transduction. For instance, does HaCat cells express CD14 and MD2. If they do not then you may need to use recombinant soluble CD14 along with LPS or pre-treat HaCat with IFN-g to increase MD2 levels then followed by LPS treatment. I hope this make sense. Good luck!!
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