I collected media in two bottles and centrifuge for 800g RCF for 10 minutes . i couldn't see any pallet , then i went for 2000g for 20 minutes still nothing what should be the possible reason?
Based on your information, it is difficult to determine what might have contributed to the outcome you noted. Therefore, one needs more information, including (i) the volume of the cell culture medium; (ii) cell-type and their growth phase; and (iii) centrifugation conditions (rotor swing bucket or fixed angle). The g-values that you noted for centrifugation of cell culture medium are not likely to "pellet" extracellular vesicles from the culture medium to the extent visible by necked eyes.
first of all, low cell number will give low exosomes. so determine the cell number. and second, for the centrifugation, the speed should be 100,000 g for at least 2 hours.
Exosomes Mediate the Cytoprotective Action of Mesenchymal Stromal Cells on Hypoxia-Induced Pulmonary Hypertension
you can refer to this paper for ultracentrifugation protocol. hope this helps.
I cultured cell in 75cm2 flask for 48hours ,90%flask was full then i treated cells with silver and DNA. Cell media was 10ml,then i thought it woud be better if i add more media so i added 15ml more then i pired media in tube to centrifuge and i coudnt see pallet
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