At 08:56 10.12.99 -0500, you wrote: >Ann- > > >>I forgot to mention that the 2nd round of sorting is after a few days in >>culture. I checked to see if its the laser bleaching, but it doesn't seem >>to be that and I can work with 80mW. >> >>The microscopy dept. is actually using the optimal filter set for CFP with >>ex. at 433nm, so if the pos. cells are faint here I guess they won't be a >>lot better at 457nm. >> >>The CFP has a minor ex.peak at 457, and a minor em peak at about 505. As I >>said the filter isn't optimal, but it worked fine for MDCK cells so I >>wasn't worried about that till now. >> >>I was afraid that CFDA might be excited at 457: The other option is single >>cell sorting from the starting sample. > >So why use CFP when so many other colors are available which are easier to >excite with, say, 488? > >-Howard > > O.K; some of the cell lines we are trying to establish will be expressing both YFP/CFP;(users are interested in visualising the secretory pathway) again some success here with another cell type. So for controls, etc, we need to get cell lines expressing each marker singly. Food for thought:::: On the double marked I sometimes see a diminished CFP signal and an increased YFP; compared to those cells in the same sample expressing only either CFP or YFP. I thought that might be a FRET signal but the users say that shouldn't happen for this particular experiment. Could it be an artifact caused by the ratio of YFP brightness (usually a good log brighter than the CFP) to CFP? i.e is the YFP casting a shadow on the CFP. Hope you can understand what I'm trying to say. I'm not worried about this for now; as when I see this I know - those are them! Ann
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