This protocol provides a feasible way to isolate and collect the right extracellular vesicles from most blood and solid tissues, especially analyze their source and the protein contents, which helps further functional experiments. Besides high yield and low contamination, the main advantage of this protocol is analysis of surface antigens and protein cargoes of EVs which is useful for physiological and pathological studies. It facilitates the diagnosis of cancers of diseases, such as inflammatory diseases and osteoporosis through the detection of surface markers of parental cells of extracellular vesicles with a flow cytometer.
The quantity of tissue extracellular vesicle is critical for the following experiments. Make sure the concentration of extracellular vesicle is not high. Be patient to perform the dilution.
To begin, prepare the maxillary bone samples by isolating the maxillary bone with ophthalmic tweezers and scissors and wash them with PBS to get rid of soft tissues with tweezers. Then put the maxillary bone into 1.5 milliliter centrifuge tubes and cut the bone into small pieces of one millimeter in diameter with scissors. Add a certain amount of Liberase to cover the tissue and incubate for 30 minutes at 37 degrees Celsius.
Next, centrifuge the sample at 800 G for 10 minutes at four degrees Celsius and carefully transfer the supernatant with a pipette to a new and clean 1.5 milliliter centrifuge tubes. After centrifuging the prepared plasma and bone samples for 15 minutes at 2, 500 G and four degree Celsius to remove large cell debris and remaining platelets, carefully transfer the supernatants to new and clean 1.5 milliliter centrifuge tubes and centrifuge at 16, 800 G for 30 minutes at four degrees Celsius. Next, discard the supernatant and resuspend the pellets in each tube with one milliliter of PBS.
After centrifuging and discarding the supernatant as demonstrated previously, again, resuspend the pellets in each tube with 50 microliters of PBS and store these samples at four degrees Celsius for less than 24 hours or preferably use them immediately for the analyses. Resuspend samples with 500 microliters of PBS and transfer 50 microliters into a new and clean 1.5 milliliter centrifuge tube as a blank control labeled as tube A and another 50 microliters into another clean 1.5 milliliter centrifuge tube as a simple staining tube for FITC, labeled as tube B.Add 0.5 microliters of membrane dye to the primary tube for membrane staining while incubating for five minutes at room temperature. After centrifuging the primary tube for 30 minutes at 16, 800 G and discarding the supernatant, resuspend the pellets with 200 microliters of PBS.
Divide each 50 microliter sample into four 1.5 milliliter centrifuge tubes for simple staining tubes for PE labeled as tube C, surface marker staining labeled as tube D, and secondary antibody-only controls labeled as tube E.Add the primary antibody of osteoclast associated receptor and bone extracellular vesicle samples as well as CD-18 antibody and plasma extracellular vesicle samples to tube B and tube D separately, incubating for one hour at four degrees Celsius. Centrifuge the tubes and discard the supernatant as demonstrated previously and resuspend the pellets with 500 microliters of PBS. Again, centrifuge for 30 minutes at 16, 800 G and four degree Celsius to remove the extra primary antibodies.
After discarding the supernatant resuspend the pellets with 50 microliters of PBS, followed by adding FITC conjugated secondary antibodies respectively in tube E.Incubate all the tubes for one hour at four degree Celsius in the dark. Dilute one drop of 0.2, 0.5, and one micrometer sized bead suspension respectively into one milliliter of PBS. Then run each size of beads to make sure of the gate chosen for extracellular vesicles and set the threshold of the flow cytometer to search for the beads and extracellular vesicle population using a suitable forward inside scatter.
Set the terminal condition as calculating 100, 000 membrane dyed particles and analyze the sample via a flow cytometer as described in the manuscript. Resuspend the pellets in 50 microliters of RIPA-lysis buffer and incubate for 30 minutes on ice. To quantify the protein concentrations of all samples in the 96 well microplate by the BCA protein assay, dilute the two milligrams per milliliters of BSA with 0.9%of normal saline into 0.5 milligrams per milliliters.
At 0.5 milligrams per milliliter of BSA and 0.9%of normal saline into three duplicated wells with certain volumes. Then drop two microliters of the samples and add 18 microliters of normal saline into three duplicated wells separately. After preparing a working solution by mixing BCA reagent A with reagent B, add 200 microliters of the working solution to each well and shake gently for 30 seconds.
Then incubate the 96 well microplate for 20 to 25 minutes at 37 degrees Celsius and measure the optical density at 596 nanometers with the spectrophotometer. Next, export the data, draw a standard curve, and calculate the protein concentration of the samples. According to the results, dilute the samples to one microgram per microliter with 0.9%of normal saline and five times the concentration of SDS-PAGE loading buffer and seal the tubes with film tightly and heat for five minutes at 100 degrees Celsius.
Load the samples in the protein ladder into a gradient concentration of four to 20%HEPy's tris gel. Run the gel in the running buffer at 80 volts until the proteins form a line. Then switch to 120 volts for one hour until the loading dye is at the bottom of the gel.
Transfer the gel to the polyvinylidene fluoride membrane pre incubated in methyl alcohol for 20 seconds using a wet transfer system to transfer for one hour at 200 milliamperes. Prepare 5%BSA blocking buffer by adding 2.5 grams of BSA and 50 milliliters of TBST into a 50 milliliter centrifuge tube. Block the membranes in this buffer for two hours at room temperature with agitation.
Incubate the membranes with specific primary antibodies diluted with TBST into proper concentration overnight at four degrees Celsius. Wash the membranes in the washing buffer four times and incubate the membranes with suitable secondary antibodies for one hour at room temperature with agitation. After washing the membranes in the washing buffer four times, image the membranes using the chemiluminescence kit in a gel imaging system.
The transmission electron microscopy and nanoparticle tracking analysis revealed that the typical morphological characteristics of extracellular vesicles were round and cup shaped with a diameter ranging from 500 to 300 nanometers. Flow cytometry analysis shows the percentages of specific membrane markers expressed on extracellular vesicles that implies their origin from parent cells. Western blot analysis shows the expression of PGD and PKM2 respectively as a representative of protein content and plasma extracellular vesicles and bone extracellular vesicles, indicating the metabolic status.
Negative expression of Golgin-84 in extracellular vesicles was detected with the presence of mitofilin, Alpha-actinin-4, Flotillin-1, Caveolin-1, and Beta-actin, along with vesicles marker CD9, while CD81 expression is low or absent with a lack of APO-A-1 existence in the plasma extracellular vesicles. Researchers, so keeping in mind that step 2.2, the preparation and the digestion of the tissue samples should be done sufficiently. Otherwise, the number of extracellular vesicles extracted from the tissues is limited.
The marker labeled fluorescent stain of the extracellular vesicles can be injected into the mouth to treat the phase that linked to potential function.
