about glucose uptake test in hepatocytes - (Oct/03/2005 )

Hello, nice to meet u all. I am a fresh postgrad and be new to this forum. I have found every different posts here are interesting and useful to my research. So I registered and logged on.

I currently have a problem with my project, which is about isolating hepatocytes followed by the detection of its glucose uptake under different drug-incubation conditions. I am currently using 3H-deoxyglucose tracer as an indiator to the degree of uptake for the test and have encountered problem in the washing step (in which cells were easily deteched from the collegan plates/ cell-bursting encountered if aggressive washings were applied, in order to get rid of the excess, non-uptaken tracer)....So are there any better ways on earth to allow a more specific counting with little cell lost?

lastly, I have heard some one before was using C14-mannitol tracer plus the deoxyglucose tracer at the same time during the test, what is the rationale for that?

Thanks for answering my question.

Kim

Kim,
How long do you leave your hepatocytes to plate before you subject them to a medium change? I work with them (I use primaria coated plates), and regardless they detach easily when you change medium. They seem to do a little better if they are allowed to proliferate longer, like 4 days.

humm....i normally do my plating of freshly prepared hepatocytes for 4 hours, on collagen-coated plates. Then right after that 4-hour-seeding period, I take my cells out and do the glucose uptake test.