外泌體基礎知識介紹，包括大小特徵、純化方法、檢測標記、儲存條件及標準化流程
Exosome size
Exosomes are small endosome derived lipid nanoparticles (30-120 nm in diameter), actively secreted by exocytosis in most living cells.
Exosome purification (BioVision)
- Chemical precipitation
- Size exclusion chromatography based
- Immunocapture based
- Filtration based exosome isolation tools
Purification result examination
You can do a western blot analysis on the isolated, purified exosomes using antibodies to exosomal markers. Just resuspend the purified exosomes in protein sample buffer (without any DTT or β-mercaptoethanol) and then blot for antibodies to exosomal markers such as:
- Alix1 protein
- CD9
- CD81
- CD63
Additionally, you can do nano tracking analysis (NTA) on the isolated exosomes.
Exosome of adipocyte
It has been shown that almost all adipocyte-derived exosomes express fatty acid binding protein (FABP4).
Exosome storage
Ideally, exosomes should be processed fresh. Fresh samples are always preferred over frozen samples.
However, -80°C frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple times(for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
DMSO-using issue
DMSO is a cryoprotectant used to preserve the cell membrane from any damage that may be caused by freezing like crystal formation for instance. However, the size of the exosomes and liquid content in it is very small as compared to the cells.
Thus, we do not recommend adding DMSO while freezing your serum samples at -80°C.
Exosome standards
Plasma exosome standards are purified lyophilized exosomes isolated from a pool of healthy certified donors by differential ultracentrifugation and microfiltration.
Exosome negitave controls
The below mentioned proteins should be absent from the Exosome preparation :
- Endoplasmic reticulum
- Grp94HSP90B1
- calnexin
(CANX) - Golgi
(GM130) - Mitochondria
(cytochrome CCYC1) - Nucleus
(histonesHIST*H*) - Argonaute/RISC complex
(AGO*)