Exosomes as biomarkers for disease Flashcards
What are extracellular vesicles?
Heterogenous group of circulating, cell derived membranous structures released mostly by eukaryotic cell types.
Where are they found?
In all bodily fluids (saliva, blood, lymph, CSF etc.)
What do they protect?
Proteins, mRNA, miRNA and lipids can be loaded either as vesicular cargo or as components of the EVs membrane. Protection allows for molecules which might be unstable or ineffective if secreted alone.
Why are exosomes different to microvesicles and apoptotic bodies?
They come from the cell surface but exosomes come from the endocytic system.
What are the steps to exosome formation?
Inward budding of the plasma membrane to incorporate surface molecules into early exosomes. Clathrin-dependent and independent mechanisms. Secondary invagination leading to formation of multiple intraluminal vesicles. Maturation into late endosome with free ILVs within a multi-vesicular body. MVBs enter lysosomal degradation pathway or fuse with PM to release cargo as exosomes into extracellular space. Small GTPases direct the traffic.
How are exosomes taken up?
Many different mechanisms look this up when less hungover.
How are exosomes isolated?
Cells are heavy and easy to get rid of so easy to form pellet at low speed. Around 500X
Spin harder to get rid of cell fragments.
Cells give off apoptotic bodies and microvesicles. Heavier then exosomes. Third centrifugation around 14000X
Put through filter
Put in ultracentrifuge at 100,000X to pellet exosomes. G-force doesn’t seem to damage them.
What is the second step to isolation?
Ultrafiltration and size exclusion chromatography. Fast and reproducible. Diluting effect so final prep has high volume and low concentration of exosomes.
What is the third step to isolation?
Exosome precipitation through polymer interaction.
How do you know exosomes are present in the sample?
Gold standard includes. TEM, nanoparticle tracking analysis (NTA) are there particles matching the expected exosome size. Cell surface marker identification by western blotting or flow cytometry, absence of contaminating cellular proteins, proteomics/ mass spectrometry.
What protein is present on exosomes?
Tetraspanning proteins. Westernblot shows this.
