To begin, centrifuge FBS overnight in an ultracentrifuge at 110, 000 G and four degrees Celsius to remove the endogenous sEVs. Sterilize the supernatant by filtering it through a 0.2 micron ultrafiltration membrane to yield sEVs-free FBS. Next in a 150 millimeter culture dish, plate about 3 times 10 to the seventh immortalized bone-marrow-derived macrophages in 20 milliliters of DMEM culture medium.
Incubate the dish at 37 degrees Celsius under 5%carbon dioxide overnight prior to collecting the sEVs from the macrophages. The next day, after discarding the medium, wash the cells with phosphate-buffered saline or PBS. Replace the medium with DMEM containing 10%sEVs-free free fetal bovine serum before incubating the cells at 37 degrees Celsius and 5%carbon dioxide.
Based on the experiment's requirements, collect and transfer the cells'supernatant to 50 milliliter centrifuge tubes. Centrifuge the tubes at 300 G for 10 minutes to remove the cells. Transfer the result in supernatant from each tube to a new 50 milliliter centrifuge tube and discard the pellet.
This time, centrifuge the supernatant at 2000 G for 10 minutes to remove dead cells. Then transfer the supernatant to new high-speed centrifuge tubes and discard the pellets. Next to remove debris and microvesicles, centrifuge the obtained supernatant at 10, 000 G for 30 minutes using a high-speed centrifuge.
Transfer the resulting supernatant to new ultracentrifuge tubes by adding 35 milliliters in each tube and discarding the pellets. Centrifuge the ultracentrifuge tubes in a swinging bucket rotor centrifuge at 110, 000 G for 70 minutes before discarding the supernatant. Wash the resulting crude sEVs-enriched pellet with one milliliter of PBS.
Again, add one milliliter of PBS to the washed pellet in one of the tubes, and mix by pipetting continuously. Transfer the one milliliter of resultant PBS suspension to another tube containing the pellet, and repeat the same until all pellets in the tubes are mixed by pipetting. Transfer the resultant one milliliter of sEVs-rich PBS to a new ultracentrifuge tube.
Then centrifuge the new tube in a tabletop ultracentrifuge at 110, 000 G for 70 minutes. After discarding the supernatant, wash the resulting crude sEVs-enriched pellet with 100 microliters of PBS. Add 1.2 milliliters of protein preparation solution in a standard protein tube to dissolve the 30 milligrams of BSA present in it.
Dilute the resulting protein standard solution with PBS to reduce its concentration from 25 to 0.5 milligrams per milliliter. Add eight different volumes of the diluted protein standard solution into wells of a 96-well plate, and bring the volume to 20 microliters in each well using PBB. Add 18 microliters of HEPES lysis buffer to the sample wells, followed by the addition of two microliters of the sEVs samples.
Next, add 200 microliters of BCA working solution prepared per the manufacturer's instructions into each well. Allow the plate to rest at room temperature for 20 to 30 minutes. Finally, measure the absorbance at 562 nanometers using a microplate reader.
Calculate the total protein content in the sEVs using the obtained results. The transmission electron microscopy images of the isolated sEVs displayed a typical cup-like morphology. Nanoparticle tracking analysis showed that the isolated sEVs were mostly concentrated at 136 nanometers.
Western blot showed that isolated sEVs were significantly enriched of sEVs markers, including CD9, beta-actin, and TSG101. The endoplasmic reticulate marker GRP94 was only detected in the whole-cell lysate. The results indicated that the method employed yielded sEVs with a high level of purity.
