We acknowledge support and advice from Dr. Jill Hutchcroft, Director of the Flow Cytometry Core Facility at Purdue University. This work was supported by R01CA226259 and R01CA205420 to A.L.K., an American Lung Association Innovation Award (ANALA2023) IA-1059916 to A.L.K., a Purdue Shared resource facility grant P30CA023168, and a flagship Fulbright Doctoral Scholarship awarded by the Department of the State, USA to H.H.

NOTE: As a prerequisite to using this technique, identification and validation of selectively exported miRNAs is required. Since different cell lines vary based on the miRNA cargo sorted into their EVs, it is recommended that the cell line of interest and associated EVs be evaluated for miRNAs prior to use. Additionally, lipofectamine-based transfection is one of the critical steps in the protocol; predetermining transfection efficiency prior to setting up the experiment is recommended.
1. Growing cells in culture and transfecting cells with fluorophore-conjugated EV-miRNA
<ol>
	<li>Plate 200,000 to 400,000 cells in triplicate wells in a 6-well plate in an appropriate culture medium. Gently swirl the plate to ensure uniform cell distribution. Incubate the cells until they reach 70%-80% confluency, approximately 24-48 h.</li>
	<li>For each well to be transfected, dilute the transfection reagent in 100&#181;L of serum-free medium in a microcentrifuge tube according to the manufacturer&#39;s instructions. 
	NOTE: The optimal concentration of lipid should be determined for the cell line of interest prior to this setup.</li>
	<li>For each well to be transfected, dilute the fluorophore-conjugated EV-miRNA (2nM) and cell-miRNA (2nM) separately in a microcentrifuge tube in 100 &#181;L of serum-free media. 
	NOTE: The total volume should be calculated for the total number of wells transfected. In this protocol, the fluorescence was evaluated at 7 time points; therefore, the total volume was 700 &#181;L for step 1.2 and 700 &#181;L for step 1.3. Additionally, to confidently capture the positive fluorescent population of cells, the user should transfect cells with a non-fluorescent scrambled miRNA and set up a gate excluding these cells.</li>
	<li>Add the diluted RNA mix (from step 1.3) to the diluted transfection reagent (from step 1.2) and mix gently by inverting the tube 3-4 times. Let the combined lipid-RNA mix incubate for 30 min at room temperature (RT).</li>
	<li>Remove the regular culture media from the cells and add 800 &#181;L of serum-free media to each well. Gently add 200 &#181;L of the lipid-RNA mix (from step 1.4) dropwise to the cells. 
	NOTE: Opti-MEM media was used for transfecting the cell line of interest (Calu6 cells). Try to add the lipid-RNA mix directly to the culture media, avoiding adding the mixture to the sides of the plate.</li>
</ol>
2. Harvesting transfected cells at various time points for analysis of fluorescence
<ol>
	<li>Incubate the transfected cells at 37 &#176;C for 7 h. 
	NOTE: To validate consistent transfection of both miRNAs (EV-miRNA and cell-miRNA), cells are collected from one well 3 h post-transfection to analyze the fluorescence using the flow cytometer.</li>
	<li>After the 7 h incubation period, remove the transfection complex from the wells and replace it with complete media.</li>
	<li>To evaluate the 7-h time-point, harvest the cells and wash the cells three times with 1x PBS.
	<ol>
		<li>If working with adherent cells, add 200 &#181;L of trypsin to one well and wait for the cells to detach from the plate. Transfer the detached cells to a microcentrifuge tube and pellet by centrifugation at 200xg for 5 min.</li>
		<li>Discard the supernatant carefully to avoid disrupting the cell pellet. For the first PBS wash, resuspend the pellet in ~500 &#181;L of 1x PBS and repeat the pelleting using the centrifugation steps above. Repeat the PBS wash an additional two times.</li>
		<li>Resuspend the cell pellet in 200 &#181;L of 2% paraformaldehyde (PFA) solution and evaluate fluorescence. 
		NOTE: After adding 2% PFA, the cell pellet can be stored at 4 &#176;C for 3-4 days. </li>
	</ol>
	</li>
	<li>Harvest the remaining time points following the steps in section 2.3. Collect all of the stored cell pellets and proceed with the steps in section 3.</li>
</ol>
3. Flow cytometry analysis of cell-miRNA and EV-miRNA signal in cells
<ol>
	<li>Prepare cells for flow cytometry analysis. Filter the cell suspension from section 2 through 35 &#181;m strainer caps that are part of the round bottom polystyrene FACS tubes to enable the removal of debris and cell aggregates that might interfere with fluorophore analysis.</li>
	<li>Set up the instrument following calibration instructions. Turn on the flow cytometer and allow it to warm up for at least 30 min before use. Prime the fluidics system with sheath fluid, and check and adjust the laser alignment.</li>
	<li>Run quality control to confirm instrument fluidics. Load ultrapure&#160;filtered water into the flow cytometer and run at an appropriate flow rate for adequate acquisition time. 
	NOTE: The appropriate flow rate and acquisition time will depend on the cell type of interest, sample complexity, and desired data quality.</li>
	<li>Open the flow cytometry data analysis software and confirm the performance of the detector and lasers. Set up threshold and voltage parameters for detection based on the cell line of interest and fluorophores of choice. 
	NOTE: For all downstream experiments, Calu6 cells were used.
	<ol>
		<li>Set the threshold for capturing the signal for Calu6 cells at FSC 5000. Perform the analysis for 1 x 104 cells for each experimental repeat.</li>
		<li>To account for the spectral overlap between different fluorophores, use un-transfected cells and cells independently transfected with only one of the fluorophores to set up compensation controls.</li>
		<li>For detection of miRNA export, use candidate EV-miRNAs (miR-451a and miR-150) conjugated to Alexa-fluor 488, and a control cell-miRNA (cel-miR-67) conjugated to Cy3.</li>
		<li>For each EV-miRNA, conjugate the fluorophore to the 5&#8242; end of the 5p strand. Before transfection, anneal the fluorophore-conjugated strand to its 100% complementary 3p strand.</li>
		<li>For the expression analysis in transfected cells, analyze the samples using the FSC, SSC, FITC, and PE channels set to 258, 192, 269, and 423 voltage units, respectively (Figure 1A, B).</li>
	</ol>
	</li>
	<li>Introduce the negative control sample into the flow cytometer. Capture cell signals after defining forward scatter (FSC) and side scatter (SSC) intensities in a custom worksheet.</li>
	<li>In the plot, choose FSC for the X-axis, SSC for the Y-axis, and gate for the population of interest by drawing a polygon around it (see Figure 1B, bottom panel).</li>
	<li>Create a new plot using the gated population.
	<ol>
		<li>Set the X-axis to fluorescence signal 1 (FS1) for cell-miRNA detection and the Y-axis to fluorescence signal 2 (FS2) for EV-miRNA detection. Set up compensation parameters for the individual fluorophores using cells independently transfected with each of the fluorescently tagged miRNAs. In the new plot, gate on the population of interest.</li>
		<li>For detection of EV-miRNA, use the FITC channel. For the evaluation of cell-miRNA signal, use the PE channel. In the software, create a gate around the cells that were positive for both FITC and PE by drawing a polygon around the double fluorescence population when FITC was selected for the X-axis and PE was selected for the Y-axis.</li>
	</ol>
	</li>
	<li>Record the statistics for the gated population.
	<ol>
		<li>Record the percentage of cells in the gated population that are positive for both FITC and PE, as well as for those that are positive for either FITC or PE.</li>
	</ol>
	</li>
	<li>Repeat the above steps for any additional samples or controls in the experiment.</li>
</ol>
4. Isolating EVs from transfected cells
<ol>
	<li>For cells cultured in serum-containing media, deplete EVs from the serum to prevent cross-contamination.
	<ol>
		<li>To generate EV-depleted serum, dilute the serum to 50% in cell culture media to reduce viscosity and centrifuge the resulting 50% serum at 110,000 x g for 18 h.</li>
		<li>Collect the supernatant (EV-free serum) carefully and filter it using a 0.2 &#181;m filter assembly. Use the resulting 50% EV-free serum to generate complete media, in this case, RPMI plus 10% serum.</li>
		<li>To collect EVs, add the EV-depleted complete media to the cells and collect the EVs 48 h after incubation. 
		NOTE: The protocol outlined below has been modified from Kowal et al.14.</li>
	</ol>
	</li>
	<li>Grow cells in 15 cm tissue culture dishes to 80% confluency. Transfect the cells separately with cell-miRNA and EV-miRNA in 10 mL of Opti-MEM following the transfection protocol in section 1 of the protocol. 
	NOTE: Not all transfection protocols scaleup linearly. It may be essential to first adapt and evaluate the transfection conditions in 15 cm plates.</li>
	<li>After 7 h, aspirate the transfection media and replace it with the EV-depleted media (15 mL). Grow cells in the EV-depleted media for 48 h.</li>
	<li>Remove the conditioned media from the cells and dispense it into a 50 mL centrifuge tube inside the biosafety cabinet. Centrifuge the conditioned media at 2000 x g for 10 min at 4 &#176;C to remove dead cells and cell debris. Transfer the resulting supernatant to a fresh tube.</li>
	<li>Centrifuge the supernatant at 10,000 x g for 40 min at 4 &#176;C. Remove the supernatant and filter it using a 0.2 &#181;m filter assembly.</li>
	<li>Transfer the filtered cell culture media to a sterile ultracentrifuge tube inside the biosafety cabinet. Weigh and balance the tubes against each other. Spin the samples at 110,000 x g for 90 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend the pellet in an appropriate volume of 1x PBS to wash and centrifuge at 110,000 x g for 90 min 4 &#176;C. 
	NOTE: The EV pellet was resuspended in 60 mL of 1x PBS based on the maximum volume allowed for the fixed-angle rotor tubes used in this study.</li>
	<li>Aspirate PBS and suspend the EV pellet in an appropriate volume (50-100 &#181;L) of fresh 1x PBS and vortex. Store EVs at -80 &#176;C for later use.</li>
</ol>
5. Flow cytometry analysis of cell-miRNA and EV-miRNA signal in EVs
<ol>
	<li>To prepare EV suspension for flow cytometry analysis, dilute EVs in an appropriate volume of ultrafiltered 1x PBS. 
	NOTE: The loading volume for the flow cytometry analysis of EVs should be determined based on the number of EVs in samples. Roughly, if there are ~30,000 particles in the EV suspension, the final volume should be ~500-600 &#181;L in 1x PBS. A clean sample has around 35-50 particles/&#181;L.</li>
	<li>Use the nano-flow cytometer or an alternative equipment powerful enough to analyze nano-sized particles. 
	NOTE: Here, Attune NxT nano-flow cytometer was used.</li>
	<li>Open the software and set the instrument up following the recommended calibration instructions. Perform the Performance Test using performance nano-beads. If the nano-beads are detected in the standard size range, the performance is optimal. Determine the appropriate flow rate for the detection of EV particles.</li>
	<li>Run quality control using ultrapure filtered water to confirm instrument fluidics and performance of detector and lasers.</li>
	<li>Using calibration beads suitable for the experiment, set the flow cytometer to accurately detect different-sized beads and measure fluorescence signals. 
	NOTE: For the setup of EV analysis by flow cytometry, ApogeeMix beads were used. These beads contain a mixture of non-fluorescent silica beads and fluorescent polystyrene beads ranging in sizes from 80 nm to 1300 nm.
	<ol>
		<li>Set up the parameters to assess the sensitivity and resolution of different populations in the bead mix according to the manufacturer&#39;s protocol. Use the appropriate gating to resolve the bead population from the instrument noise.</li>
	</ol>
	</li>
	<li>Vortex the samples thoroughly to evenly distribute EVs before loading for analysis.</li>
	<li>Open the flow cytometry data analysis software and introduce ultra-filtered PBS. Set the gating up for EV particles using ultra-filtered PBS while ensuring that the gating corresponds to the appropriate size of particles based on the beads in step 5.5.</li>
	<li>Load the negative control sample to analyze EVs as they pass through the flow cytometer. Capture EV signals while plotting FSC on the X-axis and SSC on the Y-axis. 
	NOTE: The SSC threshold for capturing EVs was set at 300. The acquisition volume was set at 95 &#181;L from a total of 145 &#181;L drawn volume. The flow rate was set at 12.5 &#181;L/min. The analysis was performed with 1 x 106 EV particles. The EV population is heterogeneous and will result in some debris isolated along with EVs. Overall particle detection in the FSC vs. SSC plot highlights particles in a diverse range, which is expected when using crude EV preparations.</li>
	<li>Create a new plot using the gated population.
	<ol>
		<li>This time, choose fluorescence signal 1 (FS1) for cell-miRNA detection on the X-axis and fluorescence signal 2 (FS2) for EV-miRNA detection on the Y-axis.</li>
		<li>Set up compensation parameters for the fluorophores using the EV samples isolated from cells transfected with a single miRNA. In the new plot, gate on the EV population of interest.</li>
		<li>For the expression analysis of fluorophore-conjugated miRNAs in EVs isolated from the transfected cells, analyze the EV samples using the FSC, SSC, BL1, and YL1 channels set to 370, 370, 400, and 400 voltage units, respectively (Figure 2A, B).</li>
		<li>Record the statistics for the gated population and visualize them using histograms and dot plots.</li>
	</ol>
	</li>
	<li>Repeat the above steps for any additional samples or controls in the experiment.</li>
</ol>
6. Validating the release of miRNAs in EVs through qRT-PCR
<ol>
	<li>Extract RNA from EVs and parent cells using miRVana RNA isolation kit following the manufacturer&#39;s instructions. 
	NOTE: Quantification of RNA isolated from EVs is not possible because the total amount of RNA isolated from EVs does not typically cross the minimal quantity threshold of a standard Nano-drop. This is due to the depletion of ribosomal RNA from EV samples, which can be validated using the Agilent Bioanalyzer. Thus, the integrity score for EV-RNA quantification is usually not reliable and is not the true representation of EV-RNA quantity. Therefore, for qRT-PCR quantification of the EV-RNA and cell-RNA, equal volumes of RNA isolates following isolation from an equal number of cells were used. For normalization of qRT-PCR, miRNAs ubiquitously present across multiple samples as determined from previously acquired RNA sequencing data were used.</li>
	<li>Convert the extracted RNA to cDNA using a reverse transcriptase kit specific for small RNAs.</li>
	<li>Set up the qRT-PCR reaction using a cDNA template and miRNA-specific primers following the manufacturer&#39;s instructions. 
	NOTE: For validation experiments, miRCURY LNA qRT PCR kit and corresponding primers from the manufacturer were used to evaluate the expression of cell-miRNA and EV-miRNA.</li>
</ol>

A Flow Cytometry Protocol for Understanding Export Mechanisms

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The authors have nothing to disclose.

The newly established protocol enables capturing kinetics of miRNA release into EVs post-transfection of EV-miRNAs. The approach allows simultaneous analysis of multiple EV-miRNAs and cell-miRNAs, subject to the capabilities of the cytometer. Moreover, while flow cytometry analysis can provide valuable insights into EV miRNA biology, it is not without limitations. Albeit some of the limitations can be overcome when used in conjunction with other techniques for a more comprehensive understanding. 
<p class="jove_conte...

MicroRNAs (miRNAs) are one of the best-characterized subsets of small noncoding RNAs, which are known for their critical role in post-transcriptional gene regulation. The expression and biogenesis of most miRNAs follow a coordinated series of events that begins with transcription of the primary miRNAs (pri-miRNAs) in the nucleus. Following nuclear processing by the microprocessor complex into precursor-miRNAs (pre-miRNAs), the pre-miRNAs are exported to the cytoplasm, where they undergo further processing by the RNase III endonuclease, dicer into 21-23 nucleotide mature miRNA duplexes1. One of the strands of the processed mature miRNA binds to target messenger RNAs (mRNAs), leading to degradation or translational repression of the targets2. Based on the pleiotropic role of miRNAs in simultaneously regulating multiple diverse target mRNAs, it is not surprising that miRNA expression is tightly regulated3. Indeed, inappropriate expression contributes to various disease states, especially in cancer. The aberrant expression of miRNAs not only represents a disease-specific signature but has also emerged as a target for prognostic and therapeutic potential4,5,6. In addition to their intracellular roles, miRNAs also have non-autonomous roles. For example, miRNAs can be selectively packaged into EVs by donor cells and exported to recipient sites where they elicit diverse phenotypic responses in normal and disease physiology7,8,9.
Despite clear evidence that EV-associated miRNAs are functional biomolecules, it is not completely understood how specific subsets of miRNAs are dysregulated in disease states such as cancer and how cellular machinery selects and sorts miRNAs into Evs10,11. Given the potential role of EV-miRNAs in modulating the microenvironment, it is critical to identify the mechanisms involved in the export of select miRNAs to fully elucidate the role of EVs in intercellular communication and disease pathogenesis. Understanding the process of miRNA release into EVs will not only highlight important mediators of miRNA export but can also provide insights into potential therapeutics. To achieve this level of knowledge, tools need to be adapted to faithfully address these new experimental questions. Indeed, studies that evaluate EVs are growing exponentially due to the introduction of new techniques and controls12,13. In relation to evaluating the abundance of exported miRNAs into EVs, next-generation sequencing and quantitative reverse transcription polymerase chain reaction (qRT-PCR) have been the current standard tools. While these tools are useful for evaluating the abundance of miRNAs in EVs, there are limitations to their sensitivity and specificity, and using these static approaches for evaluating dynamics is insufficient. Delineating the dynamics of miRNA export into EVs requires a combination of specialized tools. Here, we present a comprehensive protocol using fluorescently conjugated miRNAs, to analyze the dynamics of miRNA release from donor cells and incorporation into EVs. We also discuss the advantages and limitations of the method and provide recommendations for optimal use. The versatile method presented in this paper will be useful to researchers interested in studying miRNA release into EVs and their potential roles in cellular communication and disease.

<table><tbody><tr><td>1.5 mL microcentrifuge tubes</td><td>Fisher</td><td>05-408-129</td><td></td></tr><tr><td>15 mL Falcon tubes</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>6-well clear flat bottom surface treated tissue culture plates</td><td>Fisher Scientific</td><td>FB012927</td><td></td></tr><tr><td>ApogeeMix 25 mL, (PS80/110/500 &amp;amp; Si180/240/300/590/880/1300 nm)&amp;nbsp;</td><td>Apogee Flow Systems</td><td>1527</td><td>To set up gating and parameters for particle detection&amp;nbsp;</td></tr><tr><td>Attune Nxt Flow Cytometer</td><td>ThermoFisher Scientific</td><td>N/A</td><td>For fluorescence analysis in EVs</td></tr><tr><td>BD Fortessa Cell Analyzer</td><td>BD Biosciences</td><td>N/A</td><td>For fluorescence analysis in cells&amp;nbsp;</td></tr><tr><td>Cell culture incubator</td><td>N/A</td><td>N/A</td><td>Maintaining temperature of 37&amp;nbsp;&amp;deg;C and 5% CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Cell Culture medium</td><td>N/A</td><td>N/A</td><td>specific for the cell-line of interest</td></tr><tr><td>Cell line of interest&amp;nbsp;</td><td>N/A</td><td>N/A</td><td>Any cell line tested and evaluated for EV and cellular abundance of miRNAs on interest.</td></tr><tr><td>Cell-miRNA (miRIDIAN microRNA Mimic Red Transfection Control)</td><td>Horizon discovery</td><td>CP-004500-01-05</td><td>miRNAs predetermined to be retained by the cell-line of interest and not selectively exported out into EVs</td></tr><tr><td>EV-miRNA (Fluorophore conjugated miRNAs)</td><td>Integrated DNA Technologies (IDT)</td><td></td><td>miRNAs predetermined to be selectively sorted into EVs&amp;nbsp;</td></tr><tr><td>Fluorescence microscope</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Gibco Opti-MEM Reduced Serum Medium</td><td>Fisher Scientific</td><td>31-985-070</td><td></td></tr><tr><td>Hausser Scientific Hemocytometer</td><td>Fisher</td><td>02-671-54</td><td></td></tr><tr><td>Hyclone 1x PBS (for cell culture)</td><td>Fisher</td><td>SH30256FS</td><td></td></tr><tr><td>Lipofectamine RNAimax</td><td>Fisher Scientific</td><td>13-778-150</td><td></td></tr><tr><td>miRCURY LNA Reverse Transcriptase</td><td>Qiagen</td><td>339340</td><td></td></tr><tr><td>miRCURY LNA SYBR Green PCR</td><td>Qiagen&amp;nbsp;</td><td>339347</td><td></td></tr><tr><td>mirVana RNA Isolation</td><td>Qiagen</td><td>AM1561</td><td></td></tr><tr><td>Nuclease free water</td><td>Fisher Scientific</td><td>4387936</td><td></td></tr><tr><td>Paraformaldehyde, 4% in PBS</td><td>Fisher</td><td>AAJ61899AK</td><td></td></tr><tr><td>Reagent reservoir nonsterile</td><td>VWR</td><td>89094-684</td><td></td></tr><tr><td>Thermo ABI QuantiStudio DX Real-Time PCR</td><td>ThermoFisher Scientific</td><td>N/A</td><td></td></tr><tr><td>Trypsin 0.25%</td><td>Fisher&amp;nbsp;</td><td>SH3004201</td><td></td></tr><tr><td>Ultracentrifuge</td><td>N/A</td><td>N/A</td><td></td></tr></tbody></table>

tracking mirna release into extracellular vesicles using flow cytometry

Extracellular vesicles (EVs) are important mediators of cellular communication that are secreted by a variety of different cells. These EVs shuttle bioactive molecules, including proteins, lipids, and nucleic acids (DNA, mRNAs, microRNAs, and other noncoding RNAs), from one cell to another, leading to phenotypic consequences in the recipient cells. Of all the various EV cargo, microRNAs (miRNAs) have garnered a great deal of attention for their role in shaping the microenvironment and in educating recipient cells because of their clear dysregulation and abundance in EVs. Additional data indicates that many miRNAs are actively loaded into EVs. Despite this clear evidence, research on the dynamics of export and mechanisms of miRNA sorting is limited. Here, we provide a protocol using flow cytometry analysis of EV-miRNA that can be used to understand the dynamics of EV-miRNA loading and identify the machinery involved in miRNA export. In this protocol, miRNAs predetermined to be enriched in EVs and depleted from donor cells are conjugated to a fluorophore and transfected into the donor cells. The fluorescently tagged miRNAs are then verified for loading into EVs and depletion from cells using qRT-PCR. As both a transfection control and a tool for gating the transfected population of cells, a fluorescently labeled cellular RNA (cell-retained and EV-depleted) is included. Cells transfected with both the EV-miRNA and cell-retained-miRNA are evaluated for fluorescent signals over the course of 72 h. The fluorescence signal intensity specific for the EV-miRNAs diminishes rapidly compared to the cell-retained miRNA. Using this straightforward protocol, one could now assess the dynamics of miRNA loading and identify various factors responsible for loading miRNAs into EVs.

Here, we utilize flow cytometry as a powerful tool to investigate the release of miRNA from the cells into EVs. Using this protocol, flow cytometry analysis of cells transfected with cell-miRNA and EV-miRNA revealed a sequential decrease of fluorescence signal corresponding to EV-miRNA, while the signal corresponding to the cell-miRNA was retained in the cells. To ensure that fluorophore conjugation does not interfere with miRNA release into EVs, miR-451a was conjugated to two separate fluorophores (i.e., Alexa fluor 488...

Watch this Scientific Journal Video about Exploring the Mechanisms of MicroRNA Loading into Extracellular Vesicles in Cancer Progression at JoVE.com

Author Spotlight: Exploring the Mechanisms of MicroRNA Loading into Extracellular Vesicles in Cancer Progression 

Here, we describe a straightforward protocol that enables in vitro assessment of the abundance of fluorescently labeled microRNAs to study the dynamics of microRNA packaging and export into extracellular vesicles (EVs).

Tracking miRNA Release into Extracellular Vesicles using Flow Cytometry

Extracellular vesicles (EVs) are important mediators of cellular communication that are secreted by a variety of different cells. These EVs shuttle ...

tracking-mirna-release-into-extracellular-vesicles-using-flow

Research

JoVE Journal

Biology

1.3K Views.  Purdue University. Here, we describe a straightforward protocol that enables in vitro assessment of the abundance of fluorescently labeled microRNAs to study the dynamics of microRNA packaging and export into extracellular vesicles (EVs).

Video: Author Spotlight: Exploring the Mechanisms of MicroRNA Loading into Extracellular Vesicles in Cancer Progression 

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Genetics

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released from many cell types and play a pivotal role in regulating cell-cell communication and a diverse range of biological processes. Many different methods for the characterization of EVs have been described. However, most of these methods have the disadvantage that the preparation and characterization of the samples are very time-consuming, or it is extremely difficult to analyze specific markers of interest due to their small size and due to the lack of discrete populations. While methods for analysis of EVs have been considerably improved over the last decade, there is still no standardized method for characterization of single EVs. Here, we demonstrate a semi-automated method for characterization of single EVs by fluorescence-based nanoparticle-tracking analysis. The protocol that is presented addresses the common problem of many researchers in this field and provides the complete workflow for rapid isolation of EVs and characterization with PKH67, a general cell membrane linker, as well as with specific surface markers such as CD63, CD9, vimentin, and lysosomal-associated membrane protein 1 (LAMP-1). The presented results show a high level of reproducibility, as confirmed by other methods, such as Western blotting. In the conducted experiments, we exclusively used EVs isolated from human serum samples, but this method is also suitable for plasma or other body fluids and can be adjusted for characterization of EVs from cell culture supernatants. Irrespective of the future progress of research on EV biology, the protocol that is presented here provides a rapid and reliable method for rapid characterization of single EVs with specific markers.

In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

Biochemistry

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Immunology and Infection

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Medicine

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Size Determination and Phenotypic Analysis of Urinary Extracellular Vesicles using Flow Cytometry

Extracellular vesicles, EVs, are a heterogeneous complex of lipidic membranes, secreted by any cell type, in any fluid such as urine. EVs can be of different sizes ranging from 40-100 nm in diameter such as in exosomes to 100-1000 nm in microvesicles. They can also contain different molecules that can be used as biomarkers for the prognosis and diagnosis of many diseases. Many techniques have been developed to characterize these vesicles. One of these is flow cytometry. However, there are no existing reports to show how to quantify the concentration of EVs and differentiate them by size, along with biomarker detection. This work aims to describe a procedure for the isolation, quantification, and phenotypification of urinary extracellular vesicles, uEVs, using a conventional cytometer for the analysis without any modification to its configuration. The method&#39;s limitations include staining a maximum of four different biomarkers per sample. The method is also limited by the amount of EVs available in the sample. Despite these limitations, with this protocol and its subsequent analysis, we can obtain more information on the enrichment of EVs markers and the abundance of these vesicles present in urine samples, in diseases involving kidney and brain damage.

This protocol describes a method for the isolation of urinary extracellular vesicles, uEVs, from healthy human donors and their phenotypic characterization by the size and surface marker expression using flow cytometry.

Extracellular vesicles, EVs, are a heterogeneous complex of lipidic membranes, secreted by any cell type, in any fluid such as urine. EVs can be of ...

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular ...

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter.
Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels.
Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

The latest generation of EV characterization tools are capable of single EV analysis across multiple parameters simultaneously. Nano-flow cytometry measures all biological particles larger than 45 nm without labeling and identifies specific characteristics of subpopulations by a variety of fluorescent labeling techniques.

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and ...

Tracking miRNA Release into Extracellular Vesicles using Flow Cytometry

Summary

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Chapters in this video

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Size Determination and Phenotypic Analysis of Urinary Extracellular Vesicles using Flow Cytometry

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry