Cell Densitometry - (Jan/19/2010 )

Hi there,

I'm looking to use densitometry to analyse differences in protein expression between different groups in transfected cells that have been immunocytochemically stained. I have taken all my images at the same intensity so now I'm just looking for a way to analyse the intensity levels between the groups.

Others in my lab group have used densitometry before by analysing the first two cells on the bottom left of each image. However these studies were on cells that had been treated and so all cells in that field of view should have the same level of intensity. Here's my problem: my cells have been transfected so my images contain a mixture of transfected and untransfected cells and therefore don't have the same levels of protein intensity. For example, if I just look at the same two cells in each image, they may not have been transfected but others in the image have, so how can I accurately show changes in staining intensity.

Just wondering if anybody has any suggestions as to how I could get accurate results from these experiments. I know western blotting would obviously be the method of choice but this hasn't worked for me.

Thanks,

smr86

Try to find someone with good High Content Screening software, like Metamorph. Here if you have two different colors, one to define the boundaries of the cell and the other one that you measure within the boundary, then you can do what you want. Otherwise, you can probably use ImageJ, free from NIH. You might have to draw boundaries free hand around each cell before you analyze-measure, but eventually you will get some numbers.