This protocol is significant as it not only demonstrates approaches for purifying vesicles from culture at different scales, trade offs, differences among methodologies and also consideration for working with field samples. Cyanobacteria presents unique vesicle sampling and analysis challenges. These techniques have successfully isolated and concentrated vesicles from culture and from environmental samples.
To concentrate samples of less than 500 milliliters, start with rinsing the 15 to 20 milliliter ultrafiltration centrifugal concentrators with Type 1 ultrapure grade water. Load the concentrators into the centrifuge and spin at 4, 400 x g at four degrees Celsius. Discard the run through and repeat the step at least twice.
Load the supernatant sample into a water rinsed concentrator and spin under the same conditions. Repeat this step until the sample is concentrated to a final volume of 15 to 30 milliliters. To concentrate a volume of more than 500 milliliters, perform tangential flow filtration by setting up the apparatus as described in the manuscript.
Attach a peristaltic pump to the intake line and place adjustable clamps on the retentate line. Sanitize the tangential flow filtration as described in the manuscript, then flush the device with one liter of Type 1 ultrapure grade water. Add less than 0.2 micron filtrate into the sample reservoir.
Slowly increase pump speed and back pressure levels on the retentate line to increase output from the filtrate lines. Continue to run the tangential flow filtration and refill the reservoir with culture supernatant as the material is removed. Stop concentrating the sample once the volume in the reservoir reaches the lowest possible amount needed to maintain flow into the intake line without introducing air bubbles.
Close the outflow line with a clamp. Remove the back pressure on the retentate line. Recirculate the concentrated supernatant through the filter for 10 minutes, reducing the recirculation rate from 20 to 40 milliliters per minute to maximize the recovery.
Move the retentate line into a clean vessel, remove the intake line from the sample and collect the concentrated material. Recover any remaining material in the sample reservoir using a pipette. Filter the concentrated supernatant through a 0.2 micron syringe filter.
If needed, store the final concentrate at four degrees Celsius for approximately three weeks before moving to vesicle purification. To pellet the concentrated culture sample place it in a clean ultracentrifuge tube and add clean media or buffer to ensure that the tube is filled. After centrifugation, remove the supernatant with a pipette.
Rewash the pelleted material with fresh culture media or wash buffer, such as 1X phosphate-buffered saline and repeat the centrifugation. Resuspend the final pellet in fresh culture by gentle pipetting with a one milliliter pipette and transfer it to a clean vessel. To perform density gradient ultracentrifugation, prepare iodixanol stocks as described in the manuscript.
Mix one part of the 4X buffer with three parts of 60%iodixanol stock to make a 45%iodixanol solution. Dilute 45%iodixanol with 1X buffer to create stocks of gradient media of 40, 35, 30, 25, 20, 15 and 10%final iodixanol concentrations. Then, carefully overlay equal amounts of all these iodixanol gradient stocks into an ultracentrifuge tube, utilizing the entire tube volume.
After centrifugation, collect the fractions of 0.5 milliliters each for approximately a 4.5 milliliter gradient by carefully pipetting or using a fraction collector. Determine the density of each fraction, in grams per milliliter, using an analytical balance and calibrated pipette. Remove the sample from the tube, determine the weight and return the sample to the tube.
Dilute individual fractions with clean buffer in a new tube followed by a clean media or buffer wash. Carefully apply five microliters of vesicle and let it sit for five minutes. Remove the sample by touching the edge of a grid to a piece of clean filter paper.
Pipette out 20 to 50 microliters of 2%uranyl acetate onto a flat surface covered by plastic film. Then place the grid floating on top of it for two minutes. Remove uranyl acetate using filter paper and float briefly on a drop of ultrapure water to wash.
Fill the chamber with ultrapure water using a clean syringe and ensure no air bubbles. Click on start camera'and visualize the optimal region of the chamber around the thumbprint. Slide the screen gain'and camera level'sliders towards maximum, then decrease to the lowest level to see the dimmest particles.
Adjust the focus as necessary to ensure that particles are roughly equally visible across the field of view and continue washing with ultra clean water until the chamber is clean. Add the vesicle sample to the chamber using a one milliliter syringe and confirm the acquisition settings. Select standard measurement'from the SOP drop down box and change the settings to collect at least three technical replicates of 60 second videos each.
Press create'and run script'and follow the prompts and push approximately 100 microliters of sample into the chamber between replicates. Determine the particle parameters by clicking on analysis'and open experiment'and load the sample file. Select process selected files'and wait for the analysis to complete.
The isolation and characterization of extracellular vesicles from the cyanobacterium Synechocystis sp. PCC6803, showed that the outer membrane contains carotinoids, which confer a characteristic orange colorization to vesicle samples collected by direct pelleting through ultracentrifugation. The resuspended vesicle pellet was examined by transmission electron microscopy either by negatively staining samples with uranyl acetate or by observing ultrathin sections.
Vesicle size distribution and concentration were assessed using dynamic light scattering and nanoparticle tracking analyses. Purified Synechocystis sp. PCC6803 vesicles were separated on a sodium dodecyl sulfate-polyacrylamide gel and stained for lipopolysaccharides.
Samples should be taken before and after the concentrated vesicle sample to ensure that the tangential flow filtration is working and the vesicles are getting concentrated. Then you need to measure both samples using the nanoparticle tracking analysis. Future efforts are required to merge this method with other approaches such as the size-exclusion chromatography or asymmetric field-flow fractionation to improve the discrimination and isolation of small particles from culture and environmental samples.
This technique is helpful to study the cyanobacteria vesicles and the specific functional roles of these vesicles in cyanobacterial biology.
