Performed TEM on exosome samples isolated from cancer cell line by UC and resuspended in PBS. the majority of the image showed strange (non-exosomal) structures that could be throwing off my NTA nanosight measurements if they are from the isolation process but if they are from the TEM step i could just need new reagents.
Can anyone tell me what they are/might be and how to avoid them?
All thoughts are welcome!
Thanks
Sam
Protocol
Ultracentifugation of 48h conditioned tissue culture media (serum free)
300x g for 5 min
2000x g for 20 min
10,000x g for 45 min
100,000x g for 90 min
100,000x g for 90 min (in PBS)
Transmission Electron Microscopy
Sample - 10 min
Water - 5 sec
Uranyl Acetate - 60 sec
NB: the sample was not fixed prior to TEM
Wzosomes are fragments of cells without organelles and is certainly a new field of investigation. They are not to confound with exocytosis of known secretory products, they are pieces of cells.
Sam,
Try phosphotungstic acid as a negative stain. This has a better pH and won't precipitate in the presence of phosphate like Uranyl Acetate. 1% Phosphotungstic acid in water, adjust pH to 7.2 with a 1 N NaOH. Filter and stain as usual.
Pieces of cells that can be detected as indicated above and any other contrasting agents for TEM will work. Good luck.
If the sample was not fixed I wonder if it could be disrupted membrane fragments that are randomly aggregating (cell, exosome, apoptotic body, etc). You can also get precipitate from the UA and other elements used if they are not filtered, so be sure to do that.
As for stain, we are still trying to optimize our exosome TEM, but in my limited experience, UA is superior to PTA for both exosomes and liposomes because PTA seems to just stain these lipid vesicles as a homogeneous bright "blob," whereas UA allows you to see the "cup" shaped morphology and membrane outlines of structures.
I have attached a comparison shot of what we get with PTA vs UA.
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