Name: optimization of flow cytometric sorting parameters for high-throughput isolation and purification of small extracellular vesicles
Uploaded: 2023-01-20T14:50:00
Description: Watch this Scientific Journal Video about Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles at JoVE.com

This work was supported by the Scientific Research Fund of Zhejiang Chinese Medicine University (2020ZG29), the Basic Public Welfare Research Project of Zhejiang Province (LGF19H150006, LTGY23B070001), the Project of Zhejiang Provincial Department of Education (Y202147028) and the Project of Experimental Technology of Zhejiang University Laboratory Department (SJS201712, SYB202130).

1. Cell culture
<ol>
	<li>Prepare a culture medium of Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Culture the human pancreatic cancer cell, PANC-1, in a 75 cm2 culture flask at a density of 1 x 106 cells/mL. Incubate the culture at 37 &#176;C with 5% CO2.</li>
	<li>Subculture the cells when cell density reaches 75%-80% under microscopic observation. Remove the medium, and rinse 2x with 3-5 mL of phosphate buffer saline (PB...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology,+function,+and+biomedical+applications+of+exosomes.">The biology, function, and biomedical applications of exosomes.</a> Science. 367 (6478), (2020).</li><li>Doyle, L. M., Wang, M. 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D., Shah, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+isolating+extracellular+vesicles+impact+down-stream+analyses+of+their+cargoes.">Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.</a> Methods. 87, 3-10 (2015).</li><li>Coughlan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome+isolation+by+ultracentrifugation+and+precipitation+and+techniques+for+downstream+analyses.">Exosome isolation by ultracentrifugation and precipitation and techniques for downstream analyses.</a> Current Protocols in Cell Biology. 88 (1), 110 (2020).</li><li>Yang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=and+perspective+on+exosome+isolation+-+efforts+for+efficient+exosome-based+theranostics.">and perspective on exosome isolation - efforts for efficient exosome-based theranostics.</a> Theranostics. 10 (8), 3684-3707 (2020).</li><li>Shao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chip-based+analysis+of+exosomal+mRNA+mediating+drug+resistance+in+glioblastoma.">Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma.</a> Nature Communications. 6, 6999 (2015).</li><li>Zarovni, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+isolation+and+quantitative+analysis+of+exosome+shuttled+proteins+and+nucleic+acids+using+immunocapture+approaches.">Integrated isolation and quantitative analysis of exosome shuttled proteins and nucleic acids using immunocapture approaches.</a> Methods. 87, 46-58 (2015).</li><li>Sidhom, K., Obi, P. 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P., Duggan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+individual+extracellular+vesicles+by+flow+cytometry.">Analysis of individual extracellular vesicles by flow cytometry.</a> Methods in Molecular Biology. 1678, 79-92 (2018).</li><li>Nolan, J. P., Jones, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+platelet+vesicles+by+flow+cytometry.">Detection of platelet vesicles by flow cytometry.</a> Platelets. 28 (3), 256-262 (2017).</li><li>Dragovic, R. 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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+challenges+of+working+with+extracellular+vesicles.">Technical challenges of working with extracellular vesicles.</a> Nanoscale. 10 (3), 881-906 (2018).</li><li>Song, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+strategy+for+jet-in-air+cell+sorting+with+high+purity,+yield,+viability,+and+genome+stability.">Improved strategy for jet-in-air cell sorting with high purity, yield, viability, and genome stability.</a> FEBS Open Bio. 11 (9), 2453-2467 (2021).</li><li>Zucker, R. M., Elstein, K. H., Gershey, E. L., Massaro, E. 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This protocol outlines an optimized method to isolate and purify sEV with the specified particle size of 50-200 nm using a flow cell sorter, which was validated by NTA. The method solved the bottleneck problem of obtaining sEV with uniform particle size and high purity, avoiding interference from unrelated biological molecules wrapped in large-sized EVs22. Fast, high-throughput analyses are possible with flow cytometry, which can capture 100,000 particles per second and make 70,000 sorting decisio...

A cell releases extracellular vesicles (EVs) of varying sizes that result in signaling molecules and membrane inclusions, which are important for intercellular communication1. EVs of different sizes also play different biological roles, with 50-200 nm sEV being able to precisely distribute RNA, DNA, and proteins to the correct extracellular location. The sEV also helps determine their secretion mechanisms, involving not only the regulation of normal physiological processes such as immune surveillance, stem cell maintenance, blood coagulation, and tissue repair but also the pathology underlying several diseases such as tumor progression and metastasis2,3. Effective isolation and analysis of sEV are critical for identifying biomarkers and designing future drug interventions.
With continuous research on the clinical application of sEV, the isolation methods of sEV have put forward higher requirements. Due to the heterogeneity of sEV in size, source, and contents, as well as their similarity with other EVs in physicochemical and biochemical properties, there is no standard method for sEV isolation4,5. Currently, ultracentrifugation, size exclusion chromatography (SEC), polymer precipitation, and immunoaffinity capture are the most common sEV isolation methods6. Ultracentrifugation is still the gold standard for sEV isolation in research, despite being time-consuming, resulting in low purity with a wide size distribution of 40-500 nm and significant mechanical damage to the sEV after long-term centrifugation7,8,9. Polymer precipitation, which usually uses polyethylene glycol (PEG), suffers from unacceptable purity for subsequent functional analysis with concomitant precipitation of extracellular protein aggregates and polymer contamination10,11. Immunoaffinity capture-based methods require high-cost antibodies with varying specificity, as well as have problems with low processing volume and yields12,13,14. Particle size is one of the main indicators to evaluate the purity of isolated sEV. Although sEV purity remains an unattainable goal, SEC removes a considerable quantity of medium components, and the sEV particles extracted by the SEC method are mainly in the range of 50-200 nm15. The existing techniques have a few disadvantages, including but not limited to being time-consuming, low purity, low yield, poor reproducibility, low throughput of samples, and potential damage of sEV, which makes it incompatible with clinical utilization16. Thus, a rapid, inexpensive, and size-specified sEV isolation method applicable to diverse biofluids is an essential need in several research and clinical situations.
In flow cytometry, single particles are analyzed in a high-throughput, multiparametric manner, and subsets are sorted out17. Due to the heterogeneity of EVs, a single-particle flow cytometric measurement would be ideal, which has been used to investigate EVs following the paradigms of cell analysis, with light scatter and fluorescence labels being used to identify physiology-related features and protein components18,19,20. Nevertheless, conventional flow cytometry is challenged by the small size of sEV and low abundance of surface biomarkers. The sensitivity of flow cytometry could be improved by optimizing detection parameters to distinguish background noise and sEV regardless of using forward scatter (FSC), side scatter (SSC), or fluorescence threshold triggering parameter19.
With the present protocol, high-resolution flow cytometric sort settings were optimized using fluorescent beads as standard. By selecting the proper nozzle size, sheath fluid pressure, threshold triggering parameter, and voltages controlling scattered light intensities, we were able to isolate a specific subset of sEV from a complex mixture.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Centrifuge tube</td>
			<td>Beckman Coulter</td>
			<td>344058</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture flasks</td>
			<td>Corning&#160;</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified eagle medium</td>
			<td>Corning Cellgro</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>SUER</td>
			<td>SUER050QY</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cell sorter</td>
			<td>Beckman Coulter</td>
			<td>Moflo Astrios EQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Human pancreatic cancer cell, PANC-1</td>
			<td>NA</td>
			<td>NA</td>
			<td>PANC-1 cells were donated by Professor Weijun Yang, College of Life Sciences, Zhejiang University</td>
		</tr>
		<tr>
			<td>Laser particle size and zeta potential analyzer&#160;</td>
			<td>Malvern</td>
			<td>Zetasizer Nano ZS 90</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>Gibco</td>
			<td>C20012500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene fluorescent microspheres</td>
			<td>Beckman Coulter</td>
			<td>6602336</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission electron microscopy</td>
			<td>JEOL</td>
			<td>JEM-1200EX</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Gibco</td>
			<td>1713949</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra rainbow fluorescent particles</td>
			<td>Beckman Coulter</td>
			<td>B28479</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima-L80XP</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor</td>
			<td>Beckman Coulter</td>
			<td>SW32TI</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimization of flow cytometric sorting parameters for high-throughput isolation and purification of small extracellular vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

The flow chart diagram for the experimental protocol is shown in Figure 1. In this method, standard sized polystyrene microspheres were used as reference standards for particle size distribution. Under the specific instrumental parameter condition, the particle signal could be clearly distinguished from the background noise in the FSC vs. SSC plot using the logarithmic form. Gating strategies are shown in Figure 2. R4, R5, and R6 refer to the positions of 100 nm...

Watch this Scientific Journal Video about Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles at JoVE.com

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...

This protocol provides a rapid and size-specific solution method for small extracellular vesicles by optimizing flow cytometry sorting parameters. These optimizations provide a panel of critical sort settings that enables one to obtain representative populations of small extracellular vesicles using forward scatter versus size scatter only. Begin by performing the standard startup procedure of the flow cell sorter.Turn on the machine by pressing the startup button, then click the laser power button on the laser control panel. Press the change tanks button on the Smart Sampler submenu to pressurize the fluidics. Use an ultrasonically cleaned 50 micrometer jet and air nozzle and install it down to the nozzle assembly.Adjust the pressure to 80 SI for sheath fluid and 80.3 SI for sample flow on the pressure console. Press the start sheath flow button to start the sheath stream flowing. Click the bubble button in the Smart Sampler submenu to automatically debubble for 10 minutes and then turn it off.To align the stream and determine the laser spot, turn on the illumination chamber light to view the stream over the pinholes. Adjust the up and down, left and right, and front and back micrometers to center the stream on the pinhole and focus the stream. Open all laser shutters, select the laser and stream intercept tab and then press the green arrow button continuously as prompted to complete the laser intercept and nozzle alignment calibration process.Access the fine laser alignment screen. Select the 640 nanometer laser and 772 bar 44 bandpass channel as the x-axis parameter and the 405 nanometer laser and 448 bar 59 bandpass channel as the y-axis parameter. Press the trigger parameter selector and choose the forward scatter parameter of the 488 nanometer laser.Next, dilute the rainbow fluorescent particles by adding one drop into one milliliter of double distilled water to a final concentration of one times 10 to the sixth beads per milliliter. Open the sample chamber door and load a tube of prepared rainbow fluorescent particles. Press the start sample button and adjust up and down micrometers to optimize fluorescence intensity, making each signal as intense as possible.Press the start quality control or QC button on the touchscreen control panel to perform QC automatically. Then press the IntelliSort initialization button. The instrument automatically adjusts the frequency and amplitude of droplet oscillation to obtain the drop delay of approximately 25 and be able to visualize the break off point of the drop clearly.Next, press the drop delay button and the maintain button. Select tube as the sort output type. Place a 15 milliliter tube holder in the sort chamber.Turn the plate voltage on and set the plate voltage to approximately 4, 000 volts. Next, turn on the test pattern by selecting the stream setup button. Adjust the charge phase slider and deflection slider to ensure that the image of the stream is in line with the collection tube.Then select the check mark button. Log in to the Summit workstation on the computer. Create a new protocol from the file main menu.Then create a dot plot by selecting the histogram tab in the Summit Software control panel. Choose forward scatter as the x-axis parameter and side scatter as the y-axis parameter. Then right-click on the axis of the new dot plot in the workspace and present the forward scatter and side scatter in the logarithmic form.Select the acquisition tab and locate the acquisition parameters panel. Enable all signals in the submenu of enabling parameters. Choose forward scatter as the trigger parameter and set the threshold of 0.01.Adjust the voltage and gain of forward scatter and side scatter at 250 and 0.6 to ensure events within the forward scatter versus side scatter plot and populations are separated. Dilute the fluorescent polystyrene microspheres into 100, 200, and 300 nanometers suspensions by adding one microliter of microspheres to one milliliter of double distilled water. Then load the microsphere suspension successively.Select Start under the acquisition menu of Summit Software to record 20, 000 events and then click Stop after recording the events. Right-click on the forward scatter versus side scatter dot plot to create rectangles and drag to resize. Reposition the regions of electronic noise and 100 nanometer microsphere population by right-clicking on the regions to rename them as R7 and R4 respectively.Repeat the steps to frame the 200 and 300 nanometer microspheres with rectangles successively, and rename them as R5 and R6 respectively. Finally, determine the 50 to 200 nanometer sorting region based on the electronic noise and the position of 200 nanometer particles to define it as R8.Edit sort decisions in the sort logic and statistics panel by double clicking on the blank field of the left one or L1 stream and selecting the region named R8 to create the sort logic. Choose the abort mode of purity and select a droplet envelope above one drop for the L1 stream.Load 500 microliter sample of previously prepared extracellular vesicles or EVs mixture. Press the Start button under the acquisition menu in Summit to acquire the data and then click Stop. Next, press Start in the sort menu to collect 50 to 200 nanometer sized small extracellular vesicles or sEV into a 15 milliliter centrifuge tube.Flow cytometry analysis of standard-sized polystyrene microspheres to locate the 50 to 200 nanometer size range in the forward scatter versus side scatter plot revealed distinguishable particle signals. R4, R5, and R6 refer to the positions of 100, 200, and 300 nanometer particles respectively. R7 is the electronic noise as the detection limit for 50 nanometer particles and R8 represents the position range of 50 to 200 nanometer particles.The particle size distribution of the EVs mixture derived from pancreatic one cells by nanoparticle tracking analysis, or NTA, was in the wide range of 40 to 400 nanometers after ultra centrifugation. Flow cytometric sorting was performed to isolate and purify the 50 to 200 nanometer sEV within the R8 region. The quality of isolation was verified by NTA and it was found that the particle size range of sEV after sorting was between 50 to 200 nanometers.The presence of sEV was further observed by TEM and the isolated sEVs were confirmed to contain markers of CD6, CD63, and CD81 by Western Blot Analysis. This method allows for the separation of small extracellular vesicles and the classification or pro tem analysis of small extracellular vesicles gene expression opening up numerous downstream research applications.

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