I tested 25 ug/ml and 40ug/ml of Fluorescently labeled transferrin in melanome cells during different times of observation, but no fluorescent signal was detected inside the cells.
Is there anyone here who has had the same problem?
Thank you.
Fluorescently labeled transferring undergoes degradation after uptake by TfR. Melanoma cells are widely known to use metal ions such as copper and iron in the regulation of redox stress. Chronic iron accumulation resembles the pathophysiology of hemochromatosis or NASH, which leads to hepatic cell carcinoma, so that tumor cells depend on the aberrant redox production due to the high level of intracellular copper or iron. That is why it is likely that the fluorescent probe was degradated in the cytoplasm.
I would say that the concentration of transferrin that you are using is pretty low. Moreover, it depends on the label on the transferrin. Did you label it yourself? If that's the case, are you sure you have enough molecules of fluorophore per molecule of transferrin? What is the label? Did you try with higher concentrations? Something like 10x or even 50x what you are testing now?
Dear Go J Yoshida,
This is a good point! However, after I posted the question, I carried out the same procedure in Human Dermal Fibroblast and no fluorescence signal was detected again. So, probably, it's a trouble with my reagent.
Thank you.
Dear Marco Emanuele Favretto,
I have used transferrin in the concentration described in the literature to many cell types. The same concentration is indicated by manufacturer. I didn't label the transferrin, I purchased this reagent.
There is a chance that it be a trouble with my reagent.
Thank you for your consideration.
Katia, I can tell you that I had major problems with "commercial" labeled transferrin(s). Especially with the far red labeled ones. In order to get a decent signal, by either flow cytometry or microscopy, I had to go up to 1 mg/ml on cells overxpressing Tf receptor... this is way I am suggesting to increase the concentration and see what you get. Just from personal experience.
Marco Emanuele Favretto,
on second thought, I'll carry out a test with higher concentrations. Personal experience are better than description on articles.
After, I tell you the result.
Thank you.
Marco Emanuele Favretto,
on more question: do you treat the cells with some special buffer or medium?
I have used serum free RPMI or a buffer indicated by manufacturer (HEPES, BSA, Glucose).
Thank you.
Hi Katia,
sorry for the late reply. I use serum free RPMI for short term incubation (max 1 hour) and switch to a low percentage serum (1-5%) for longer incubation times.
Keep us updated
Hi Kátia Morais ,
I'm coming a bit late but I also have trouble with transferrin, I can't detect any signal after using the concentration given by the literature. Did you try to use it at a higher concentration on the HDF cells? Do you remember if it worked ?
I do thank you
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