The overall goal of the following experiment is to determine whether cells and biological fluid of interest release or contain RNA containing exosomes. The first step to isolate exosomes is to remove cells and cell debris. Next, the supernat is then filtered to remove larger particles.
The exosomes are then pelleted through ultracentrifugation as a second step. The exosomes are characterized by electron microscopy, flow cytometry, and western blood analysis to show that the isolated vesicles have the morphology size and protein composition of exosomes. Next RNA is isolated from the purified exosomes.
In order to determine which RNA, the exosomes contain the results that are obtained by these well-established methods for purification detection and characterization of exosomes can facilitate further studies of exosomes such as their biological functions. So this presentation will show you how exosomes can be isolated through different means, and the two people that will present that are my two PhD students, Maria, El and Cecelia. Er.Good luck.
The human mass cell line, HC one is used for this exosome isolation demonstration to begin the procedure to isolate exosomes. First, grow cells in medium with exosome free serum. Then transfer the cell suspension to conical tubes and centrifuge at 300 times G for 10 minutes at four degrees Celsius.
To pellet the cells transfer the super name to ultra centrifuge tubes and centrifuge the sample at 16, 500 times G for 20 minutes at four degrees Celsius to further remove cells and cell debris. Next, filter the SANE through a 0.2 micron filter to further remove particles larger than 200 nanometers. Finally, the ultracentrifuge tubes containing the filtrated sane is sealed an ultracentrifuge at 120, 000 times G for 70 minutes at four degrees Celsius to pellet the exosomes.
The exosomes are retrieved by cutting open the ultracentrifuge tube, discarding the snat and resus suspending the exosome enriched pellet. For maximal exosome retrieval. Resus suspend the exosome enriched pellet repeatedly in a small volume of an appropriate buffer into an eend orph tube.
Rinse twice with additional buffer of the same volume as previous. The buffer depends on the type of experiments following the exosome.Isolation. For electron microscopy use approximately 10 micrograms of exo somal protein from the intact exosomes.
Resuspended in PBS performed. Our carbon coated nickel grid electron microscopy can be performed without immunostaining as exosomes can be identified solely based on size morphology. However, it is recommended to use a primary antibody such as anti CD 63 or anti MHC Class two, followed by 10 nanometer gold labeled secondary antibodies.
As exosomes are too small to be detected by current flow cytometry equipment, it is necessary to first bind the exosomes to antibody coated beads. Depending on the exo somal cellular origin, different antibodies are coupled to magnetic or latex beads. In this demonstration, the anti CD 63 coated latex beads will be used for each sample.
Use a volume equal to 30 to 40 micrograms of exo somal protein of the intact exosome. Suspended in PBS incubate exosomes in beads overnight at four degrees Celsius under gentle movement. The next day blocked by adding 300 microliters of 200 millimolar glycine and incubate for 30 minutes.
Wash the exosome bead complex is twice and wash buffer incubate the exosome bead complexes with 50 microliters IgG antibody at four degrees Celsius. Wash the exosome bead complexes twice, add 90 microliters, wash buffer, and 10 microliters antibody of choice to the exome bead complexes and incubate for 40 minutes in the dark. Under gentle movement, wash the exome bead complexes twice, add 300 microliters wash buffer, and transfer the samples to flow cytometry tubes and acquire data using flow cytometry.
This western blot demonstration will focus on the importance of a suitable protein isolation before the Western blot and the different antibodies used. Dissolve the exosome pellet in the protein lysis puff of choice and pipette thoroughly followed by vortex mixing to further ly the exosomes, sonicate the sample in a water bath centrifuge the sample 13, 000 times G for five minutes and transfer the SANE to a new einor tube. Measure the total protein by a method of choice and load the protein on the gel.
Then separate and transfer the proteins by gel electrophoresis and electro blotting block and wash the membrane before performing immunostaining against proteins enriched in exosomes as there are no exosome specific markers. Proteins that are enriched in exosomes from all different cellular origins are commonly used for exosome detection. These are proteins such as tetraspanins, for example, CD nine, CD 63, and CD 81.
Cytoskeleton associated proteins such as ezrin and proteins involved in multivesicular biogenesis such as TSG 1 0 1 and LY as a negative control check for contaminating proteins commonly found in vesicles from other compartments such as kexin and GRP 78 from the endoplasmic reticulum. Different RNA isolation kits can be used depending on planned experiments. In this demonstration, a column-based method will be used when performing RNA isolation.
It is important to use nucleic acid and nuclease free pipette tips and wipe both bench and equipment free from contaminants and RNAs. Dissolve the exosome pellet in 350 microliters of lysis solution and vortex the sample. Add 200 microliters of 95%ethanol and mix using a vortex.
Place a column in a collection tube and transfer the lysed exosomes to the column and centrifuge Wash three times in 400 microliters of wash solution to make sure that the column is dry. Centrifuge for two minutes at 14, 000 times G after the last wash and place the dry column in an RNAs free eend rph tube elute. The RNA in 50 microliters elucian buffer for detection and analysis of the extracted total exo somal, RNA.
An Agilent 2100 bioanalyzer can be used with the RNA 6, 000 nano or RNA 6, 000 pico kit as exosomes are too small to be detected by available flow cytometry methods. They're attached to antibody coated beads before they're analyzed. As these exosome bead complexes can aggregate a flow cytometry, scatterplot can contain different populations of single double and triple exosome bead complexes.
As shown here, the arrow indicates the population of single exosome bead complexes, which is the population that is further analyzed. A flow cytometry histogram shows a single exosome bead population positive for the CD 63 protein. The further to the right that the open curve is, the more positive the sample is for CD 63.
Due to the small size of exosomes, they can only be visualized with electron microscopy. Typical morphological characteristics of exosomes include round shaped 30 to 100 nanometer size ous vesicles. An exosome immuno stain with gold liberal antibody for CD 63 is shown indicated by the Arrow Western blot.
Results show the absence of the endoplasmic reticulum protein calnexin in the exo omal sample, but show the presence of the tetraspan in CD 81, which is a protein commonly enriched in exosomes. Cellular RNA mainly contains ribosomal RNA, seen as the two peaks for the 18 s ribosomal RNA subunit seen on the left and the 28 s subunit seen on the right in this bioanalyzer analysis. Exo somal RNAs, however differ in their profiles as they contain little or no ribosomal RNA and are enriched in short, RNA such as messenger, RNA and micro RNA Once mastered.
The isolation of can be performed in approximately three hours if done correctly. While using this method, it's important to perform all the steps, including the non-filing to avoid contaminating of larger ics. After development of this technique, it has become much more easy to characterize exosomes, both biochemically as well as biologically.
Furthermore, we've learned much more about the exosome mediated cell to cell communication in different systems. Also, the technique has made it possible to develop exosomes as biomarkers for different diseases. Thank you very much for your attention.
