Hi!
I'll need to isolate RNA and protein from mouse hepatocytes. I don't need to culture them, so I was wondering if there is any way to isolate hepatocytes without any perfusion step? I would like to cut off a small part of the liver and then digest it with a collagenase solution. Would this be possible?
Thanks a lot!!
If you don't to treat hepatocytes nor culture them you can just simply isolate RNA from liver tissue. Hepatocytes is the most relevant cell type.
You can also try to digest a piece of liver but you will obtain less vital cells.
Good luck
Thank you both for your suggestions! The problem I have is that I need to check a hepatocyte specific downregulation of a gene, so I want to avoid any RNA from different cell types (even though hepatocytes are the most relevant cells in the liver). I was thinking of some possible centrifuge steps?
Dear Max: Are these hepatocytes you wish to isolate RNA from a specific treatment group ( you mentioned down regulation, I am assuming in response to some exposure, potentially in vivo)? Or are you simply using an untreated source? What strain? Are you using previously perfused and cryo-preserved cells as your system? I can put you in contact with our expert teams here to discuss both an off the shelf starting product (with better hepatocyte purity) and some insights to cell based RNA isolation that we use everyday here at XenoTech. Best of luck.
Whether you collagenase digest your livers or you mash them through a screen, if you resuspend your cells in a large volume (like 40 mL) and then allow settling time, or do a slow spin (1-2 min at 30 x g), what settles out will be hepatocytes. Most other cell types will remain in the supernatant. You can't expect to get 100% purity out of the settled cells, but it's a good start.
Leonardo, how did you performed your experiment? I'm asking because I'm in a similar situation, but I need to stain the cells. I am thinking about what Micelle suggested. I would appreciate if you help me!
Well, actually... i also met the same situation, i needed to sort out the some hepatocytes with fluorescence to extract the RNA.. i did not use the classical two-step perfusion protocols(Could somebody professional tell the necessities and significance of perfusion step by the way..Coz i really don't know why we cannot directly cut into pieces and digest the liver)..i just digested the liver with Collagenase V and Dnase I, then used the FACS to sort out the cells.... the DAPI staining showed the good viability of cells