Other Protocols
Primary hepatocytes are highly versatile due to their high metabolic activity and diverse functions in vivo; many, if not all of these functions may be reproduced in vitro. Glycogen synthesis, glucose output, beta oxidation, lipogenesis, and xenobiotic metabolism (EROD) are only a few of the functional assays we have extensively tested and validated in these cells. In addition, we have broad experience with standard biochemical and molecular assays (e.g. Western blotting and qRT-PCR); the overwhelming majority of these assays are hormone-sensitive and/or otherwise manipulable with agonists/antagonists.
For now, I have provided static PDFs of a few select protocols that are sufficiently complete for public distribution. I hope to present these methods in more interactive HTML format in the near future, along with custom-made data processing spreadsheets that should significantly facilitate data analysis. There is a spreadsheet included for the beta oxidation assay below, which I feel somewhat comfortable sharing due to inclusion of comments in most cells that should provide enough information to be understood.
Please pay close attention to the date stamps on these protocols. Updated versions, when provided, will have more recent time stamps. This page will be updated in the near future when I have polished up my remaining protocols and spreadsheet tools.
Glycogen and gluconeogenesis protocols
Contains general procedures for assessing glycogen synthesis, glycogenolysis, and gluconeogenesis in primary hepatocytes.
Digestion of glycogen in primary hepatocytes
Procedure for lysing cells and purifying and digesting glycogen from plated primary hepatocytes
Glucose oxidase-peroxidase protocol
Protocol for measurement of glucose from media, purified/digested glycogen, and a host of other sample sources. A much lower-cost, easy-to-make, very easy to use alternative to expensive kits.
Tritium-labeled long-chain fatty acid beta oxidation in primary hepatocytes
A protocol for beta oxidation using tritium-labeled palmitate. Applicable to other long-chain fatty acids that undergo beta oxidation. Contains procedures for coupling FFA to BSA as well. This spreadsheet provides a semi-automated calculator that suggests volumes of radiolabel, concentrations, etc. to expedite experiment design and minimize radioactive waste; see comments in cells for instructions. Note that this sheet is protected, but may be unprotected without a password. Please use with Excel 2007 or above-- may require conversion for Mac. A static (formula-free) example is included with some additional notes for guidance. This example is representative of what one would print out for bench use.
EROD protocol in plated, frozen primary hepatocytes
A streamlined protocol for the EROD assay, optimized for frozen hepatocytes, using a spectrofluorimeter. Contains detailed information for making resorufin standards.
ProQuest-published thesis
The published version of my thesis work. A somewhat lengthy document that contains a relatively up-to-date summary of my hepatocyte-related work. Can also be downloaded directly from the ProQuest site (open-source).