The goal is to study endocytosis. Dextran uptake was done at 37C for 3 hours, 5 hours or overnight using a concentration of 1 or 5 mg/ml; I either get no signal or a diffuse signal throughout the cell. Does anyone have a suggestion for the dextran uptake? [NB: transferrin or antibody uptake worked fine.]
The most likely reason is that your dextran prep has a lot of free dye, a sign of insufficient clean up after fluorescent labeling. You could either contact your vendor to obtain properly prepared dextran or dialyzes it with PBS multiple times (remember to demand your money back!).
I agree with Dr. Zha, but you should also use a larger dextran since the 10 kDa material may be lost upon dialysis. Dialysis will work better than a short sizing column because the free rhodamine does associate weakly with the rhodamine dextran, so a slower procedure is actually more complete.
A lot depends on whether you are fixing and permeabilising the cells at the end of the dextran uptake period and prior to looking at them by microscopy or flow cytometry. If you fix and permeabilise then you are likely to loose most of the staining. There are lysine fixable Dextrans out there but we have only had limited success with them in fixed cell analysis.
By performing live cell imaging we see very nice dextran labelling including 10KDa- TRITC Dextran. If you just fix and leave out the permeabilisation step then you are also more likely to see nice staining but we have not tried this with 10KDa- TRITC.
Many questions arise about your exact setting. Dextran can be taken up by very different mechanisms including 1) receptor-dependent clathrin-mediated endocytosis (CME) via MR, DC-SIGN family receptors and langerin, 2) macropinocytosis or fluid phase endocytosis (FPE), 3) Cdc42-dependent endocytosis (in rare settings), 4) probably by others not yet well-understood mechanisms. Mechanism of uptake will depend on the cell type. So the cell type, receptor expression and activation state are all of a big importance as well as a type of dextran. Dextran-10 is poorly taken up by CME because it does not bind much of receptors. You could try using FD35 or FD70.
Dextrans are used mainly for macropinocytosis/fluid phase endocytosis and it causes the diffuse signal. It should happen within minutes. Transferrin labels different types of endosomes such as clathrin dependent endocytosis. Thatswhy transferrin works for you but not dextran. Dextran-40 is ideal.
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