Exosome Block Preparation: A Protocol to Preserve Exosomes From Cultured Human Colon Cancer Cells
Trascrizione
Exosomes are the extracellular vesicular structures that are secreted by cultured cells into the supernatant. To prepare an exosome block, begin by taking an exosome-containing culture supernatant in a tube. Centrifuge the tube at high speed to pelletize the intact exosomes. Discard the remaining supernatant.
Treat the exosome pellet with glutaraldehyde present in the fixing buffer. Incubate the mix on ice for the desired time to preserve the morphology of exosomes. Glutaraldehyde infiltrates the exosomes and crosslinks the proteins. Fixing buffer maintains exosomes at neutral pH for the downstream fixation process.
Discard the glutaraldehyde-containing buffer and add osmium tetroxide solution – a secondary fixative that permeabilizes the membrane bilayer and fixes the lipids present in exosomes. Remove the osmium tetroxide solution and treat the exosomes with a series of increasing concentrations of acetone. This process dehydrates the exosomes.
Replace absolute acetone with the embedding medium-acetone mixture. Gradually, increase the ratio of embedding medium in this mixture.
Pour the embedding mixture containing exosomes into desired molds. Oven-dry the molds at a high temperature. This will lead to polymerization of the embedding medium around the exosomes, giving rise to blocks.
