Dear All I am new to the world of flow cytometery, a technique that I think can be applied to answer the following experimental questions. Although, I am familiar with fluorescence imaging of epithelial cells, I have not previously worked with a prokaryotic/eukaryotic co-culture system. I anticipate incubating cultured colon epithelial cells with a broth culture of gut microflora for up to 24 hours. At the end of the culture period I would like to analyse gut epithelial cell cycle and cell number, and intercellular and adherent bacterial cell number. After removing the broth at the end of the experiment I, therefore, need to distinguish three cell populations: gut cells with surface bound bacteria, gut epithelial cells with phagocytosed bacteria, gut cells that are not associated with bacteria. Preferably, this would include labelling of gut epithelial cell DNA for cell cycle analysis. In the short term it is possible to label the bacteria broth culture with a SYTO or CFDA-SE dye etc. from Molecular Probes and then incubate them together with gut epithelial cells. This will enable us to quantify bateria-epithelial cell adhesion. Ideally, I would like to specifically label the cell types at the end of the experiment so as not to interfere with their physiology. Unfortunately, the fluorescent dyes known to me that label bacteria nuleic acids, for example, also label gut epithelial cells. An alternative approach would be to label the bacteria with a suitable bacteria-specific antibody, but I cannot think of a conserved target antigen since the gut broth culture contains a mixture of diverse bacteria. I was hoping that you may have some suggestions as to how I may be able to distinguish the three cell types that I have outlined above. It is the lack of a specific bacterial fluorescence label that appears to be the problem and I can only hope that you know of one. Alternatively, this may involve a combination of familiar fluorescent dyes, forward scatter and side scatter that I have failed to recognize. Dr. Mark Williams School of Biological Sciences University of East Anglia Norwich Norfolk NR4 7TJ http//www.uea.ac.uk/~b289/ Tel 01603 592244 Fax 01603 592250 e-mail M.R.WILLIAMS@uea.ac.uk WWW Server URL: http://www.bio.uea.ac.uk
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