This simple protocol allows one to label EVs and monitor them via confocal microscopy. For instance, EV imaging might help in evaluating EV distribution when use it in therapeutics. This technique allows one to monitor EV migration and uptake in cartilage explants easily, with confocal microscopy without any special function.
The same protocol can be used for EVs for in vitro or in vivo experiments. Make sure that the columns are prepared beforehand, and that the initial volume of EVs is enough to reach the desired final particle concentration, considering that around 10%of the particles will be lost during the purification step. Begin by concentrating the extracellular vesicle, or EV, sample and the control.
Place the samples in a 15 milliliter or 500 microliters concentrating tube, depending on the starting volume of the EV sample. Centrifuge the tubes according to the manufacturer's instructions until an almost dry sample is obtained. Re-suspend the concentrated EV samples with 200 microliters, and the control group with 100 microliters of diluent C, and transfer them to new 1.5-milliliter centrifuge tubes.
Separate the EV sample into two aliquots of 100 microliters each. Mark one of the aliquots with dye and use it as treatment. Leave the other unmarked, but process it like the EV sample, and use it to quantify the EV concentration by nanoparticle tracking analysis.
Prepare 2x dye solution such that the resulting concentration of PKH26 solution in diluent C is eight micromolar. Mix one microliter of one-millimolar PKH26 linker per 125 microliters of diluent C in the sample. Prepare the volume required to add to the samples in a 1:1 ratio.
Add 2x dye solution to PKH platelet lysate-derived EVs and control samples in a 1:1 ratio, to achieve 1x dye concentration, and four micromolar PKH26 concentration. Add the same volume of PBS to the NTA platelet lysate-derived EV sample. Incubate for five minutes at room temperature.
Add 5%bovine serum albumin-PBS solution to the samples in a 1:1 ratio, and ensure that the final volume is approximately 400 microliters. Proceed to separate the labeled EVs from the unbound dye and non-specific dye interactions with the column. Remove the cap from the column, add 400 microliters of PKH platelet lysate-derived EVs, NTA platelet lysate-derived EVs, or control and discard all eluted liquid.
Wait for the sample to enter the column completely before proceeding to the next step. Add 600 microliters of PBS and discard all eluted liquid. Wait for the PBS to enter the column completely before proceeding to the next step.
Add 600 microliters of PBS and collect a fraction of 600 microliters in a 1.5-milliliter centrifuge tube. For a new column, repeat the steps involving the concentration of the samples mentioned at the beginning of the protocol. Add 600 microliters of previously eluted EVs to the column and discard the eluted volume.
Then add 400 microliters of PBS and discard all eluted volumes. Add 600 microliters of PBS to the column and collect a fraction of 600 microliters in a 1.5-milliliter centrifuge tube. Wash the cartilage two times with PBS and excise it using a three-millimeter diameter biopsy punch under sterile conditions.
Place the explants in 96-well culture plates with DMEM-F12 medium, supplemented with 1%penicillin streptomycin at 37 degrees Celsius, 5%CO2, and 80%humidity. To establish an inflammation-driven OA model, supplement the cell culture medium with 10 nanograms per milliliter of oncostatin M and two nanograms per milliliter of tumor necrosis factor-alpha. Treat the explants with 1 times 10 to the 9th particles per well of PKH platelet lysate-derived EVs, or control, in cell culture medium supplemented with oncostatin M and tumor necrosis factor-alpha.
Remove the medium from the 96-well cell culture plates containing cartilage explants. Add 200 microliters of the cell culture medium to each well as described in the manuscript. Stop the in vitro assay every hour from zero to five hours.
Wash the cell culture wells containing the cartilage explants two times with 200 microliters of PBS. Add 100 microliters of 4%paraformaldehyde to the tissue to fix it for three hours at four degrees Celsius. Remove the PFA, add 100 microliters of PBS, store the fixed tissue at four degrees Celsius, and process the samples within 48 hours.
Images taken at different time points show how EVs enter the tissue until they reach the chondrocytes, and enter the chondrocytes over time. The labeled EVs were already localized around chondrocytes after one hour of incubation. Ensure that the initial volume of EVs is enough to reach the desired final particle concentration.
Another important thing to remember is to use the labeled EVs within the next 24 hours. If not use it within 24 hours, a decrease in fluorescence may occur. This technique will help researchers to improve EV imaging, and to study EVs in biology and therapeutics.
