I have isolated exosomes from conditioned cell culture supernatants, but have not witnessed any pellet after two steps of ultracentrifugation. Is it normal or should I must see some pellets?
Any suggestions?
Pellets from cell media can be difficult to see. Usually, I will only see the faintest indication of a pellet. It's more of a difference in refractive index (the light bends differently through the pellet). But anyways, yes this is normal. Keep track of the outer edge of the tube and you should be fine.
Thank you Matt Jackson and Madhurima Chatterjee
for your answers. I have seen that people isolating exosomes from serum and other body fluids usually get pellet, so I was wondering whether I should also get it. Do you have experience of isolating exosomes from serum or any body fluids?
Yes, from plasma. There will indeed be a pellet. Ultra from plasma is wrought with coprecipitated proteins, etc. My pellets are brownish, the color of plasma, and very difficult to resuspend or lyse. Same goes for PEG precipitation, even if I preceed with a proteinase K digestion.
Dear Matt Jackson , why is it likely to get pellet in case of plasma but not in case of cell culture supernatants? Is it because in conditioned media the exosomes concentration is less?
Plasma is much more complex than conditioned media. Macromolecules are abundant, and the pellet is far from pure. See Arraud et al. https://onlinelibrary.wiley.com/doi/full/10.1111/jth.12554
There is little agreement in the literature concerning the concentration of EVs in plasma, with values ranging from 200 to 109 EVs μL−1 50, 66. Low EV concentrations have been consistently obtained by conventional FCM, which is in agreement with our finding that FCM detects only a minority of EVs. On the other hand, EV concentrations up to 109 EVs μL−1 PFP have been reported by NTA 50, 67, hence several orders of magnitude larger than our EM results. It is likely that non‐vesicular particles like lipoproteins or macromolecular complexes contribute to these large values, as shown by Dragovic et al. 50.
Sasmita Samal Pre-concentrate your culture medium (e.g. using 100KDa ultrafiltration tube) before ultracentrifugation have higher chance to give you a visible pellet. Or, if you are using differential centrifugation, instead of denstiy gradient, you may also consider to perform a parallel control experiment by spiking in fluorescent label liposome with similar size range, which might resulted in a coloured pellet.
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