This method can help address questions related to the efficiency, recovery yield, and purity of bronchoalveolar lavage fluid derived extracellular vesicles using an ultrafiltration centrifugation approach. We directly compared this technique to an ultracentrifugation and density gradient purification technique. The main advantage of this technique is that it is simple, efficient, and suitable for small volume samples such as murine bronchoalveolar lavage fluid.
More importantly it provides high purity and significantly higher scalability compared to an ultracentrifugation isolation method. Demonstrating the procedure will be Mr.Norman Garrett III, research associate for my laboratory. To begin, insert a 22 gauge angiocatheter into the trachea of a euthanized mouse.
Attach an insulin syringe with one milliliter of ice cold sterile DPBS, and instill one milliliter into both of the lungs. Slowly withdraw the syringe plunger to retrieve bronchoalveolar lavage fluid, or BAL fluid. And dispense the BAL fluid into a conical tube on ice.
After this, pull BAL fluid from 25 mice and divide it into two equal sets. Centrifuge the BAL fluid at 400 times G for five minutes at four degrees celsius. Collect the supernatant and centrifuge it at 15, 000 times G at four degrees celsius for 10 minutes.
Then collect the supernatant and proceed to the EV isolation steps immediately. First, filter the super natant through a 0.2 micrometer sterile syringe filter. After this, equilibrate the centrifugal filter unit with sterile DPBS for 10 minutes.
Then centrifuge the centrifugal unit at 15, 000 times G for 10 minutes at four degrees celsius. Fill the filter with 15 milliliters of BAL fluid. And centrifuge it at 3, 000 G's for 30 minutes at four degrees celsius.
After this, wash the retentate with 14 milliliters of sterile DPBS by gently pipetting repeatedly. Centrifuge the filter unit at 3, 000 G's at four degrees celsius for 30 minutes to remove the DPBS and concentrate the retentate. To collect the concentrated BAL fluid derived EV's, insert a pipetter into the bottom of the filter unit, and withdraw the sample using a side to side sweeping motion.
Then aliquot the BAL fluid derived EV's and store them at minus 80 degrees celsius. Alternatively, start by transferring the previous acquired supernatant to an ultracentrifuge tube. And centrifuge it at 10, 000 G's for 30 minutes at four degrees celsius.
Then collect the supernatant and centrifuge. Discard the supernatant and re-suspend the EV pellet in 200 microliters of DPBS. After this, mix the EV suspension with 300 microliters of 50%iodixanol working solution.
And transfer it to a 15 milliliter ultracentrifuge tube. Then add buffer solution according to the text protocol to create a buoyant density gradient, and centrifuge it at 100, 000 G's for three hours and 50 minutes. Collect the 15%and the 25%iodixanol fractions, and dilute them in DPBS to bring the volume up to 15 milliliters.
Transfer the fractions to a new ultracentrifuge tube and centrifuge at 100, 000 G's at four degrees celsius for 60 minutes. Discard the supernatant and re-suspend the EV pellets in 50 microliters of sterile DPBS. To begin nano particle tracking analysis, dilute the EV sample in one milliliter of DPBS.
And load the sample into an insulin syringe. After this, attach the syringe to a syringe pump, and measure the particle numbers and size. Set the camera level to 14 and the detection threshold to one.
Next, use a plate reader to quantify the amount of protein in the EV sample via colorimetric detection. Dissolve an equal amount of EV protein from each sample with a blotting loading buffer and DTT. Then heat the samples at 70 degrees celsius for 10 minutes.
Load the samples into a Bis-Tris Plus acrylamide gel and run electrophoresis for 35 minutes. Then transfer the proteins to a nitrocellulose membrane using a dry transfer method. After this, block the membrane with five percent skimmed milk for 60 minutes with gentle rocking.
Incubate the membrane with an antibody to an EV surface protein marker at four degrees celsius with gentle rocking overnight. Develop the membrane by incubating the membrane with HRP link antibody for 60 minutes at room temperature. Wash the membrane for 10 minutes with TBST buffer three times.
Finally, develop the membrane with chemiluminescent HRP antibody detection reagent before proceeding to imaging. To begin flow cytometry, dilute BAL fluid derived EV's in 49 microliters of PEB staining buffer. Then add three antibodies to each of the samples, before incubating the samples at four degrees celsius with rocking for 60 minutes in the dark.
Finally, dilute the samples with 40 microliters of membrane filtered PEB staining buffer and perform flow cytometry analysis. In this protocol, EV's were isolated from mouse BAL fluid using a UFC and UC-DGC methods. BAL fluid derived EV's from normal mice isolated by the UFC method displayed smaller sizes and more uniform size distribution than the UC-DGC-EV's.
The UFC technique also had significantly higher total particle counts when compared to the UC-DGC technique. The total protein recovery measured in milligrams of the UFC-EV's was also higher than that of the UC-DGC-EV's, indicating that the UFC technique is more time and effort efficient. Finally, the BAL fluid derived EV's were also further characterized via flow cytometry.
The UFC-EV's and UC-DGC-EV's both expressed CD63, CD9, and CD81. While attempting this procedure, it's important to remember to rehydrate the ultrafiltration unit membrane with DPBS. Once the membrane is hydrated, the unit needs to be kept wet at all time until being used.
Retrieving extracellular vesicle retentate from the filter unit can be challenging. Make sure that the pipette tip is swept side to side to recover most of the EV's from the filter unit. After its development, this technique paved the way for researchers in the field of extracellular vesicle isolation to explore the biological functions of bronchoalveolar lavage derived extracellular vesicles in both normal or pathological conditions in small animals.
