Problem:
In response to my previous question, I've been reading up a little bit on poly-D-lysine, Collagen I, Collagen IV, laminin, and other tissue culture coatings that promote cell adhesion. I've always assumed that anything other than standard TC-treated plastic or glass would significantly increase background, but perhaps my views on background fluorescence are a little outdated.
Question: Does anybody have experience with these in a fluorescent microscopy/high-throughput screening environment?please give your suggestions.
Specifically in my case, I'm looking at endocytosis and trafficking of a labeled protein into the lysosome. I'm labeling the protein with the pH-dependent dye pHrodoTM from Molecular Probes, which supposedly has very little fluorescence at neutral pH, but becomes very bright as the pH drops when endocytic vesicles become lysosomes. This theoretically means that a final wash step is not needed, but with a matrix coating on the plates I'm worried about background.
Question: what is the current thinking as far as background fluorescence of the various TC matrices is concerned? Describe briefly.
Question: Does the background come from the matrix itself, or by the fluorescent dye becoming adsorbed to it? Is it wavelength-dependent? Explain in detail.
Fortunately I may not be stuck with my poorly-adhering cells, and I may not need supplemental matrix at all in the end, but I still want a better understanding of how it works.