mouse hepatocytes isolation

[ramya313]

hi!
is there an antibody or a marker that i can use to be certain that the cells i have isolated are indeed murine hepatocytes?

[ramya313]

hi!
i am new to isolating hepatocytes, however i think i've managed liver perfusion through the portal vein and have been able to isolate hepatocytes. My problem is that i dont know if they are hepatocytes! after a day of isolation, i can see some binucleated cells and they eventually spread out but the other cells in the same well look round but are still attached. I donno what these round cells are and i donno if they are non parenchymal cells or any other contaminating cell population.
I wanna know if anybody knows of any murine hepatocyte surface markers that will help me confirm!
thanks

[NickW]

Here is a paper where they characterized their hepatocyte cell isolation by immunohistochemistry (see materials and methods)

Journal: Liver International
Author: Bryan L. Copple
Date: 2010
Title: Hypoxia stimulates hepatocyte epithelial to mesenchymal trasnsition by hypoxia-inducible factor and transforming growth factor-B-dependent mechanisms

Nick

[ramya313]

Hi Nick
Thanks a ton for that paper, I've one more favour to ask of you: I was reading through the thread and i found your detailed protocol for isolation of hepatocytes, but i could not access the figures and id love to see them. I'm new to the forum so could you pls tell me how to access?if not, is there another way to see em?
ramya

[NickW]

Ramya,
The protocol posted does not have pictures.

I have a pdf file with some pictures of liver perfusion and hepatocyte isolation in a rat. I don't think i can upload the file here. It is a hepatocyte isolation protocol from a biotech company, AMAXA. I can no longer find the file online. i could email it to you.


below are some links that might help. [Only registered users see links. ]

[Only registered users see links. ]

[NickW]

Ramya
do Google video seach for "hepatocyte isolation". There are actually videos on YouTube. i have not watched them, but they are there. Maybe it will help.
Nick

[wr_1a]

thanks

[samona1]

Has anyone ever tried hepatocyte isolation via perfusion of the spleen?

I would also like to isolate primary hcs and have been told this way was much easier...

Thanks!

[TamH]

Would it be possible to dissociate the liver without perfusion if I only need to analyse the cells by FACS? For example by just incubating for a longer time in collagenase and cutting the liver up more with a scalpell or so?

[angeTHEmuse]

@TamH: what kind of FACS markers are you going to use on those Hepatocytes?