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Transferrin-alexa fluor633 can not be endocytosed by Vero cells. Who knows why? - (Jan/02/2008 )

In my experiments, transferrin-alexa fluor633(TF633) is used as an example for clathrin-mediated endocytosis. After Vero cells are washed three times with PBS and then incubated for 1h in opti-mem supplemented with 0.2% BSA at 37℃, TF633 is added at a final concentration of 10ug/ml. But when the cells are observed under a leica confocal microscope and excited with a 633nm laser, no fluorescent signal appears on any cell. It seems that TF633 tends towards being absorbed to the cover slide instead of the cells. Any advice will be appreciated!


Check the following possibilities: 1) cells should be seeded less than conflent. 2) use plane DMEM. Opti-MEM may have transferrin in it. 3) optional: do a FITC-dextran co-uptake experiment for pinoccytosis. 4) for your question with TF633 binding to cover slide, did you coat the cover slide with PLL? 5) Although not the best choice for high magnification (40X), regular tissue culture plate is fine for many applications (under 20X).


Have you tried other labelled Tf? Best to use either FITC- or rhodamine-Tf first, so that you can SEE (without the need for confocal), because the draw-back of the Far-Red dyes is that you cannot see them well by eye, so for the first time usage, you can't be sure if the abnormal results are real or not.
Have you used this particular Tf before? In our experience, for Far-Red labelling, the Tf-AF647 works very much better than the 633.


Many thanks to genehunter-1 and almasy for your precious advices.
I think I have found my answer.


Replacing opti-mem with plane DMEM solved my problem.