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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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 ...

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