The overall goal of the following experiment is to quantify and size profile extracellular vesicles using tunable resistive pulse sensing. The standard approach is to compare current blockade measurements taken from purified extracellular vesicles with those taken from polystyrene bead standards. A second approach to characterizing extracellular vesicles is to spike the sample with polystyrene beads, which is used when working with complex samples such as un purified extracellular vesicles.
In cell culture medium, the resulting data characterizes the size and number of the extracellular vesicles, whether they are purified or un purified. The main advantage of this technique over existing methods like western blotting or flow cytometry of ex extracellular vesicles bound to latex beads, is that this method characterizes particles directly in biological fluids without the need for physical isolation or labeling. Generally, individuals new to the method will struggle because extracellular vesicles are heterogeneous in size.
When trying to detect the smaller vesicles, larger vesicles may clog the nanopore. We have added some practical tips and tricks to the protocol to quickly return to workable conditions. We tried to reduce the production of extracellular vesicles and needed a methods to quantify the concentration of vesicles in cell culture.Snet.
In search for methods to characterize nanos particles, we came across TRPS and modified its protocols to be more suitable for characterization of sles and cell culture. S supernatants, and other biological fluids Begin by connecting the TRPS instrument to a computer with the eyes on control suite software installed on it. Be sure to minimize electrical interference as discussed in the text protocol.
Now, choose the Nanopore size. An NP 100 is the best choice for 70 to 200 nanometer vesicles, whereas an NP 150 can be used for 85 to 300 nanometer sized vesicles. An NP 200 is better suited to measure 100 to 400 nanometer vesicles.
Then select the complementary polystyrene calibration particles for the NP 100 and NP 150 select CPC 100 calibration particles for the NP 200. Use CPC 200 calibration particles. Vortex the calibration particles for 30 seconds.
If they are still aggregated, use sonication then dilute the calibration particles in PBS to the target concentration. Based on the nanopore size, the final volume must be at least 40 microliters. Now wet the lower fluid cell of the instrument by applying 78 microliters of PBS and immediately removing it.
This reduces the risk of bubbles forming under the nanopore. Next, place the Nanopore into the for arms of the instrument. Measure the distance between the two opposite arms using digital calipers.
Input this value into the software under the field called stretch and once entered, click on calibrate stretch using the side wheel that controls the distance between the opposing arms. Stretch the nanopore to 47 millimeters. Once stretched to size, reapply 78 microliters of PBS to the lower fluid cell.
Begin the calibration process by placing the upper fluid cell onto the nanopore and attaching the shielding cage. Then add 40 microliters of diluted calibration particles into the upper fluid cell. Now apply at least 0.8 kilopascals of positive pressure.
Using the variable pressure module or VPM, select a positive voltage and click turn on. Then slowly reduce the stretch while analyzing the blockade. Events caused by the calibration particles.
As the stretch decreases, the blockade height will gradually increase and the signal to noise ratio will thus improve. Increasing the voltage can also increase the blockade height, but can also generate more RMS noise. Stop reducing the stretch when the observed blockades appropriately surpass background levels as noted from the signal trace panel.
Secondly, observe the particle rate. However, this has a less strict cutoff. The particle rate should ideally be at least 100 per minute.
However, if the particle rate is greater than 2000 per minute, dilute the sample and recalibrate the instrument. A rate this high can lead to inaccurate measurements for this demonstration. Previously purified extracellular vesicles from a brain tumor cell line are characterized.
Start by loading the calibration particles into the upper fluid cell press. Turn on then apply pressure using the VPM and record at least 500 particles of data here. A pressure of 0.8 kilopascal is applied.
Optionally, a multi pressure measurement can also be made to do this, increase the applied pressure and record a second calibration file. The pressure increments must be at least 0.2 kilopascals now, remove the sample from the upper cell and wash the cell three times with 100 microliters of PBS per wash. After the washes, wipe off the upper fluid cell with lint-free paper.
Add the experimental sample next. The baseline current must be within 3%of the current observed when measuring the calibration particles. If not, tap or twist the shielding cap, apply the plunger or completely remove the nanopore and wash it down with di water.
If the observed current is good, apply the exact same pressures as applied to the calibration particles. Then record at least 500 particles and save the data file. If there is a sudden interruption of particle detection, a drop in baseline current or a sudden increase in RMS noise, then pause the recording and try to unclog the nanopore.
As before, pipette the sample up and down. Tap or twist the shielding cap, apply the plunger or completely remove the nanopore and wash it down with DI water. Another strategy is to increase the nanopore stretch up to 47 millimeters and maximize the pressure from the VPM for about five minutes.
As this can also UNC unplug the NPO in the software. Go to the analyze data tab and start processing the data by right clicking the unprocessed files option and select the process files option. Then to couple the sample and calibration files, click the checkbox next to the sample.
In the calibrated column, select the corresponding files and okay. The choices. The software will display different sample characteristics like size, distribution, baseline durations, full width, half maximums, and concentration analytics.
The following approach is used for samples such as biological fluids that lead to excessive clogging using the standard method in preparation, centrifuge at least 50 microliters of cell culture. Supernatant containing the extracellular vesicles for seven minutes at 300 Gs.Also prepare a media alone control in the same way for both the sample and control. Transfer 20 microliters of the supernatant to new tubes and add 20 microliters of PBS and 10 microliters of diluted 335 nanometer polystyrene bead stock at 10 million beads per milliliter.
Now set up the instrument as before, using an NP 200 Nanopore and measure the control sample of just beads in media. First, for accuracy, the background detection of small non bead particles must be minimized to less than 10%of the bead detections. Now, measure each sample once before recording replicates.
This will distribute the fluctuating nanopore conditions over the samples. Each sample should be measured three times in all finish by re measuring the calibration bead only sample.Later. During data analysis, use spreadsheet software to make concentration calculations.
An example calculation is included in the written protocol. Extracellular vesicles were purified from U 87 M-G-E-G-F-R-V three cell culture, supernatant by ultracentrifugation, and then measured using the described protocol using 115 nanometer calibration beads. A size distribution for the extracellular vesicles was obtained.
Extracellular vesicles were also quantified directly from glioblastoma cell culture supernatant, using the alternative approach that spikes the sample with a standard. Two nanoparticle populations were observed. The smaller extracellular vesicles and larger known standard, the size estimation of the two populations based on the known standard, identified the vesicle population as greater than 140 nanometers.
Smaller extracellular vesicles may have been detected using a smaller nanopore opening, but there would have been more clogging Once mastered. This technique can be done in one to four hours depending on the number of samples analyzed. While performing this procedure, it's important to remember that samples may require septal adjustments to the protocol.
For instance, samples consisting of larger vesicles may require the usage of larger nanopores at relatively large stretch, and also measurements can be facilitated by filtering our centrifugation of samples to remove cellular debris, which can clock the nanopore. After watching this video, you should have a good understanding of how to characterize extracellular vesicles using TRPS. Depending on your extracellular vesicle sample, you can apply the standard protocol or use the adjusted spike in method.
