I am fractionating conditioned media and I am performing sequential centrifugation in order to separate out apoptotic bodies, microvesicles and exosomes. I am wondering whether it would be possible to use a histology dye to stain the cell membrane so I know where my pellet is and how substantial it is.
P.S. I will need to perform DNA extraction on these fractions.
You can stain them, but exosomes are to small to be recognized by light microscopy. IMHO
You may find the following article helpful. It refers to DNA extraction after use of histological dyes. They state which dyes can be used before extraction and which have negative effects.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1907345/
(some more references here: https://books.google.com/books?id=KBvgUFZ3yeMC&pg=PA63&lpg=PA63&dq=histology+dye+dna+extraction&source=bl&ots=5gRALhHzYq&sig=5LEuKbvrO5APikDz5_fwUoMtBD8&hl=en&sa=X&ei=eBX_VPWiB4eqyQTftYKgBw&ved=0CGkQ6AEwCA#v=onepage&q=histology%20dye%20dna%20extraction&f=false )
After your last centrifuge you can see the pellet, for sure, but you can't distinguish an exosomes pellet from an apoptotic bodies pellet, for example.
Currently we don't have a list of EV-specific ‘‘markers’’ that distinguish subsets of EVs from each other. Analytic approaches can include western blots (WB), flow cytometry (FACS), global proteomic analysis using mass spectrometry techniques.
Principally, at least 2 different technologies should be used to characterize individual EVs: Electron microscopy and Atomic force microscopy.
For larger vesicles such as apoptotic bodies you can use Cytospins and Immunofluorescent images.
For size distribution you can use nanosight, Dynamic light scattering and Resistive pulse sensing.
I am also very interested in this as I also have a small amount of cells, which I need to pellet to be able to lyse them properly. Did you already found a solution Robert?
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA