Hello dears,
I will thank you for your answers,
I want to culture hepatocyte in Williams medium E, but I am not sure how I do. after the culture how I can resuspend this cells. Do you have a technique?
To evaluate gluconeogenesis and Glycogenolysis, I want to culture with DMEN medium without L-Glutamine. How would it affect my assays?
We culture primary hepatocytes in DMEM-low glucose with 10% FBS. L-glutamine is not essential for gluconeogenesis, but we always added. However, it may leads to a failure with some compounds when you perform an assay.
Besides, just after isolation, we will culture hepatocytes in HepatoZYME-SFM(Gibco 17705021) adding L-glutaMAX and 10% FBS, which will enhance cell adherence and livability. And before assay, cells will be serum starved for at lest 6 hours. This is not essential too, depending on your budget.
Good luck!
Thanks @Yang Lou. I would like to ask. How can i separate after culture?. can I use any enzyme for that.
Er. sorry i forgot it. I thought you would culture hepatocytes for glycogenolysis and gluconeogenesis at first, and we don't resuspend them in the two assay.
Of course you can use enzymes such as trypsin for 30 s. However, then hepatocytes may not be able to attach to plates or wells anymore.
Ok, thank you very very much Yang Lou. you are the best person in the world. So I don´t need enzymes because I am going to work glycogenolysis and gluconeogenesis.
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