RE: Flow cytometry in Rat Primary Hepatocytes

Sir -

According to the age of your rats, you can expect to see as many as 3 different populations based upon ploidy and cell size. These populations are 2n, 4n and 8n populations. Within the 4n and 8n populations you will see mononucleate and binucleate populations. The functionality of these populations may vary according to the function you are measuring.

Ronald K. Charlton, Ph.D. 
Director, Transplant Immunology Laboratory 
Shands Jacksonville Medical Center 

-----Original Message-----
From: Kruti.Naik@sanofi-aventis.com [mailto:Kruti.Naik@sanofi-aventis.com]
Sent: Wednesday, March 23, 2005 9:23 AM
To: cyto-inbox
Subject: Flow cytometry in Rat Primary Hepatocytes



Hello, 

I am currently working on a project involved in detection of Reactive Oxygen Species (ROS) in Rat primary hepatocytes. In our lab we actually established a method for detection of ROS in a rat secondary hepatoma cell line, which worked very well however we are having some troubles with optimizing the conditions in the primary hepatocytes. The particular instrument we are using is FACSCalibur from BD biosciences. We are using superoxide-specific dye Dihydroethidium (HE) and another dye Sytox Green (SG) for staining the dead cell population. With rat primary hepatocytes the problem I am having is that I am getting three different live populations but only one of them is giving me a shift in fluorescence upon ROS production and also it is dose-dependent. My question is how can I be sure that the population I am looking at is a hepatocyte population. Also this particular population is coming up in the 10(2)  to 10(3) on the log scale in FL2 channel specific for HE. Should I be concerned about that and try to shift this further down on the log scale so as to see a more drastic shift? We also thought about sorting these different populations and trying to observe them under the microscope to make sure that we are looking at hepatocytes and not anything else. Also is it to use the sorted cells and run them on the flow again or are the cells pretty much useless after sorting?

Would really appreciate any possible help. 

Thanks a lot in advance, 

Kruti Naik. 

Kruti Naik 
Sanofi-Aventis Pharmaceuticals, 
Bridgewater, NJ. 
USA.