Denise, I have a protocol for mouse hepatocyte isolation (not the easiest of tasks by the way). I will attach a detailed protocol and also my recent publication using a technique. Also this protocol can be found on my website: www.renabahjat.com under the protocols link. A few hints: I would recommend finding a good surgical microscope or surgical loops (even a magnifying light will help) for inserting the catheter into the inferior cava, as I could not do it by eye in the mouse. Make sure to throw a stitch around the catheter as the flow rate used will cause it to slip out. A big problem to watch out for is that after isolation you need to spin the cells at a relatively low speed 150g, then wash with media but be VERY CAREFUL in resuspending them. Gently rocking the conical tube works even though it takes forever. NEVER VORTEX or you will not recover a pellet upon centrifugation. I found this out the hard way. As for flow cytometry, hepatocytes do not survive the process, particularly after isolation. Hepatocytes are VERY fragile once removed via collagenase perfusion, but can recover when plated overnight. Although, I still have not been able to get them to survive flow cytometry but you can give it a try. Let me know if you have problems as I have even made a video tape of the microscopy procedure (insertion of the catheter and perfusion of liver) for posterity and I would be happy to provide it to you. Also, using collagenase works well if you find a good lot. If you plan to make a habit of doing this protocol it would be worth the time investment. However, blendzyme from Roche works more consistently or atleast was easier to troubleshoot than the different lots of collagenase. I have felt your pain...... Reference: Bahjat et al., Journal of Immunology see the following website: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_ui ds=11086099&dopt=Abstract Rena Bahjat Graduate Assistant University of Florida College of Medicine Department of Surgery Research Laboratory websites: http://www.renabahjat.com or http://www.surgery.ufl.edu/html/divisions/research/index.htm phone: 352-265-0494 fax: 352-265-0676 address: PO Box 100286 Gainesville, Florida 32607 ---------------------------------------------- "The test of a first-rate intelligence is the ability to hold two opposed ideas at the same time, and still retain the ability to function." --F. Scott Fitzgerald, 1896-1940 -----Original Message----- From: Manfra, Denise [mailto:denise.manfra@spcorp.com] Sent: Tuesday, February 12, 2002 12:54 PM To: cyto-inbox Subject: mouse hepatocyte isolation Hi: Does anyone have a protocol for isolating hepatocytes from mouse liver. We have tried a couple with little success. We would like to use them for culture and for FACs. Thanks, denise *************************************************************** This message and any attachments is solely for the intended recipient. If you are not the intended recipient, disclosure, copying, use, or distribution of the information included in this message is prohibited -- please immediately and permanently delete this message. <<Murine hep isol and plating.doc>> <<mouse diag.jpg>>
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:26:00 EST