We thank Emanuele Canonico for technical support. Part of this work was carried out in ALEMBIC, an advanced microscopy laboratory established by IRCCS Ospedale San Raffaele and Universit&#224; Vita-Salute San Raffaele. The work of Enrico Ragni and Laura de&#160;Girolamo was supported by the Italian Ministry of Health, &#34;Ricerca Corrente&#34;.

Fluorescence-Based Sorting of Adipose-Derived Extracellular Vesicles

<ol>
	<li>Welsh, J. A., 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=Minimal+information+for+studies+of+extracellular+vesicles+(misev2023):+From+basic+to+advanced+approaches.">Minimal information for studies of extracellular vesicles (misev2023): From basic to advanced approaches.</a> J Extracell Vesicles. 13 (2), e12404 (2024).</li><li>Xu, R., Greening, D. W., Zhu, H. J., Takahashi, N., Simpson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle+isolation+and+characterization:+Toward+clinical+application.">Extracellular vesicle isolation and characterization: Toward clinical application.</a> J Clin Invest. 126 (4), 1152-1162 (2016).</li><li>Dixson, A. C., Dawson, T. R., Di Vizio, D., Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context-specific+regulation+of+extracellular+vesicle+biogenesis+and+cargo+selection.">Context-specific regulation of extracellular vesicle biogenesis and cargo selection.</a> Nat Rev Mol Cell Biol. 24 (7), 454-476 (2023).</li><li>Witwer, K. W., Wolfram, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+versus+synthetic+nanoparticles+for+drug+delivery.">Extracellular vesicles versus synthetic nanoparticles for drug delivery.</a> Nat Rev Mater. 6 (2), 103-106 (2021).</li><li>Cheng, L., Hill, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutically+harnessing+extracellular+vesicles.">Therapeutically harnessing extracellular vesicles.</a> Nat Rev Drug Discov. 21 (5), 379-399 (2022).</li><li>Du, S., 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=Extracellular+vesicles:+A+rising+star+for+therapeutics+and+drug+delivery.">Extracellular vesicles: A rising star for therapeutics and drug delivery.</a> J Nanobiotechnology. 21 (1), 231 (2023).</li><li>Keshtkar, S., Azarpira, N., Ghahremani, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell-derived+extracellular+vesicles:+Novel+frontiers+in+regenerative+medicine.">Mesenchymal stem cell-derived extracellular vesicles: Novel frontiers in regenerative medicine.</a> Stem Cell Res Ther. 9 (1), 63 (2018).</li><li>Orbay, H., Tobita, M., Mizuno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+isolated+from+adipose+and+other+tissues:+Basic+biological+properties+and+clinical+applications.">Mesenchymal stem cells isolated from adipose and other tissues: Basic biological properties and clinical applications.</a> Stem Cells Int. 2012, 461718 (2012).</li><li>Caplan, A. I., Dennis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+as+trophic+mediators.">Mesenchymal stem cells as trophic mediators.</a> J Cell Biochem. 98 (5), 1076-1084 (2006).</li><li>Rani, S., Ryan, A. E., Griffin, M. D., Ritter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell-derived+extracellular+vesicles:+Toward+cell-free+therapeutic+applications.">Mesenchymal stem cell-derived extracellular vesicles: Toward cell-free therapeutic applications.</a> Mol Ther. 23 (5), 812-823 (2015).</li><li>Galipeau, J., Sens&#233;b&#233;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells:+Clinical+challenges+and+therapeutic+opportunities.">Mesenchymal stromal cells: Clinical challenges and therapeutic opportunities.</a> Cell Stem Cell. 22 (6), 824-833 (2018).</li><li>Jia, Y., 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=Small+extracellular+vesicles+isolation+and+separation:+Current+techniques,+pending+questions+and+clinical+applications.">Small extracellular vesicles isolation and separation: Current techniques, pending questions and clinical applications.</a> Theranostics. 12 (15), 6548-6575 (2022).</li><li>Ohnuma, K., Yomo, T., Asashima, M., Kaneko, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorting+of+cells+of+the+same+size,+shape,+and+cell+cycle+stage+for+a+single+cell+level+assay+without+staining.">Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining.</a> BMC Cell Biol. 7, 25 (2006).</li><li>Morales-Kastresana, A., 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=High-fidelity+detection+and+sorting+of+nanoscale+vesicles+in+viral+disease+and+cancer.">High-fidelity detection and sorting of nanoscale vesicles in viral disease and cancer.</a> J Extracell Vesicles. 8 (1), 1597603 (2019).</li><li>Mortati, L., 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=In+vitro+study+of+extracellular+vesicles+migration+in+cartilage-derived+osteoarthritis+samples+using+real-time+quantitative+multimodal+nonlinear+optics+imaging.">In vitro study of extracellular vesicles migration in cartilage-derived osteoarthritis samples using real-time quantitative multimodal nonlinear optics imaging.</a> Pharmaceutics. 12 (8), 734 (2020).</li><li>Andreu, Z., Yanez-Mo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetraspanins+in+extracellular+vesicle+formation+and+function.">Tetraspanins in extracellular vesicle formation and function.</a> Front Immunol. 5, 442 (2014).</li><li>Mildmay-White, A., Khan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+markers+on+adipose-derived+stem+cells:+A+systematic+review.">Cell surface markers on adipose-derived stem cells: A systematic review.</a> Curr Stem Cell Res Ther. 12 (6), 484-492 (2017).</li><li>Welsh, J. A., Tang, V. A., Van Der Pol, E., Gorgens, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MIFlowCyt-EV:+The+next+chapter+in+the+reporting+and+reliability+of+single+extracellular+vesicle+flow+cytometry+experiments.">MIFlowCyt-EV: The next chapter in the reporting and reliability of single extracellular vesicle flow cytometry experiments.</a> Cytometry A. 99 (4), 365-368 (2021).</li><li>Maia, J., 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=Employing+flow+cytometry+to+extracellular+vesicles+sample+microvolume+analysis+and+quality+control.">Employing flow cytometry to extracellular vesicles sample microvolume analysis and quality control.</a> Front Cell Dev Biol. 8, 593750 (2020).</li></ol>

The authors have nothing to disclose.

Analyzing and sorting EVs is challenging due to their small size and the fact that they are near the detection limit of most flow cytometers. Our objective was to develop a protocol for isolating EVs derived from AMSCs labeled with CFSE. CFSE was selected as the staining method due to its reported high EVs labeling efficiency (&#8805;90%), without the formation of unwanted particles such as protein aggregates given by antibodies. Nevertheless, it is possible that few EVs without esterase could be missed and future studie...

Extracellular Vesicles (EVs) are a heterogeneous group of membrane-structured vesicles released by almost all cells, delimited by a lipid bilayer, unable to replicate on their own1. They can be found in several biofluids such as blood plasma, serum, saliva, breast milk, urine, bronchial lavage fluid, amniotic fluid, cerebrospinal fluid, and malignant ascites2. One of the main functions of EVs is to transport various molecules, including nucleic acids, proteins, lipids, and carbohydrates, between a donor and a recipient cell. This can occur through various mechanisms, such as direct membrane fusion, receptor-ligand interaction, endocytosis, and phagocytosis3,4. For this reason, they have been demonstrated to play an important role in a lot of physiological and pathological processes, and they show considerable promise as novel biomarkers of disease, as drug delivery vehicles, and as therapeutic agents5,6.
Mesenchymal stromal cells (MSCs) are multipotent cells that can be isolated from many tissues, including adipose tissue, dental pulp, umbilical cord blood, placenta, amniotic fluid, Wharton&#39;s jelly, and even the brain, lung, thymus, pancreas, spleen, liver, and kidney. In recent years, they have attracted considerable interest in regenerative medicine7. Adipose-derived mesenchymal stem cells (ASCs) can be harvested from fat tissue through a less invasive procedure compared to other sources like bone marrow, resulting in lower risks of severe complications and avoiding ethical issues8.
Additionally, adipose tissue contains a significantly higher concentration of MSCs than bone marrow (1% versus &#62;0.01%) and other sources such as the dermis, dental pulp, umbilical cord, and placenta. MSCs are crucial in the regeneration of injured tissues and cells due to their differentiation ability and their secretion of a broad repertoire of growth factors, chemokines, and cytokines; these therapeutic benefits are attributable to their differentiation ability but also to the fact that they secrete a broad repertoire of growth factors, chemokines, and cytokines. A striking example is given by MSCs&#39; therapeutic potential for orthopedic conditions, with the term &#34;Musculoskeletal Diseases&#34; having the higher number of registered clinical studies under clinicaltrials.gov (accessed 13th May 2024).
Moreover, MSCs can also secrete EVs that take part in tissue regeneration via transferring information to damaged cells or tissue and exert biological activity similar to the mother cells9,10. For this reason, MSC-EVs may be a valuable substitute for cell therapy to achieve a cell-free approach11, with two clinical studies involving MSC-EVs for orthopedic conditions (NCT05261360 and NCT04998058). However, several challenges still exist for the clinical applications of EVs. For example, there are some concerns about EV isolation techniques: most of them do not guarantee vesicle purity or integrity. Moreover, some isolation techniques are complex, time-consuming, and have low repeatability, making them unsuitable for clinical use12.
Cell sorting, on the other hand, is a commonly used method that allows for the isolation of single cells from heterogeneous cell suspensions by using specific fluorescent markers13. It can be used for many applications and adapted to different sample types. However, although cell sorting is a well-established and widely used technology, EV sorting is still very challenging because most EVs are below the minimum detection threshold for even the most sensitive flow cytometers. There are some features that make a sorter more suitable for this purpose. First of all, using a Jet-in-air system in which the stream suspending the particles is interrogated by lasers in air, rather than within a flow cell; this system preserves the sample by decreasing the stress to which it is subjected. A second important point is the presence of an &#34;obscuration&#34; bar between the stream and the collection lens that decrease the background optical noise of the instrument. Although it is low, the background noise is not completely eliminated and constitutes a reference that provides a partial window into the events that fall under the threshold: it is very important for the analysis of events that are close to the &#34;limit of detection&#34; of the instrument14. Finally, the sorter features a dual-path Forward Scatter (FSC) with two different masks that allow for improved discrimination between small and large particles in the sample.
Based on this, we developed a protocol aimed to separate carboxyfluorescein succinimidyl ester (CFSE) labeled MSC-EVs by using a high-sensitivity cell sorter. To minimize the manipulation of EVs and preserve their integrity and quantity, we avoided ultracentrifugation steps during the sample preparation. Furthermore, sorting conditions were adjusted to minimize stress on the vesicles, including further optimization of our instrument by reducing the sorting pressure associated with the nozzle size (70 &#956;m nozzle for a pressure of 35 psi).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5(6)-Carboxyfluorescein diacetate&#160;N-succinimidyl ester</td>
			<td>Merck</td>
			<td>150347-59-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Adipose Mesenchymal Stromal Cells</td>
			<td>Wepredic, Parc d&#39;affaires, 35760 Saint-Gr&#233;goire, France</td>
			<td></td>
			<td>Cells used in this study</td>
		</tr>
		<tr>
			<td>Alexa 488 anti-Caveolin</td>
			<td>R&#38;D Systems</td>
			<td>IC5736G</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>APC anti-human CD44</td>
			<td>BioLegend</td>
			<td>338805</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>APC anti-human CD63</td>
			<td>BioLegend</td>
			<td>353007</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>APC anti-human CD81 (TAPA-1)</td>
			<td>BioLegend</td>
			<td>349509</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>APC anti-human CD9</td>
			<td>BioLegend</td>
			<td>312107</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>BC CytoFLEX S</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>BC CytoFLEX S equipped with 3 lasers, Blue, Red and Violet</td>
		</tr>
		<tr>
			<td>Flow-Check Pro Fluorospheres</td>
			<td>Beckman Coulter</td>
			<td>A63493</td>
			<td>Fluorescent control beads for MoFLO Astrios EQ</td>
		</tr>
		<tr>
			<td>FlowJo software (version 10.8.1)</td>
			<td>BD</td>
			<td>version 10.8.1</td>
			<td>Analysis software</td>
		</tr>
		<tr>
			<td>IntraSure kit</td>
			<td>BD Biosciences</td>
			<td>641776</td>
			<td>Fixation and permeabilization for intracellular staining</td>
		</tr>
		<tr>
			<td>Megamix-Plus FSC</td>
			<td>BioCytex</td>
			<td>7802</td>
			<td>FSC polystyrene beads&#160;</td>
		</tr>
		<tr>
			<td>MoFLO Astrios EQ</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>MoFLO Astrios EQ equipped with 4 lasers, Blue, Yellow - Green, Violet and Red</td>
		</tr>
		<tr>
			<td>Mouse anti-FLOT1 antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610820</td>
			<td>Western Blot antibody</td>
		</tr>
		<tr>
			<td>NanoSight NS300</td>
			<td>Malvern</td>
			<td>NS300</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-Calnexin antibody</td>
			<td>Origene</td>
			<td>TA336279</td>
			<td>Western Blot antibody</td>
		</tr>
		<tr>
			<td>Rabbit anti-CD9 and CD81 antibody (ExoAb antibody kit)</td>
			<td>System Biosciences</td>
			<td>EXOAB-KIT-1</td>
			<td>Western Blot antibodies</td>
		</tr>
		<tr>
			<td>Rabbit anti-TSG101 antibody&#160;</td>
			<td>Merck</td>
			<td>HPA006161</td>
			<td>Western Blot antibody</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Merck</td>
			<td>9036-19-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Rainbow Fluorescent Particles</td>
			<td>Spherotech</td>
			<td>URFP-30-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracel 100 kDa MWCO</td>
			<td>Merck</td>
			<td>UFC910024</td>
			<td></td>
		</tr>
		<tr>
			<td>VER01 - Verity Shells</td>
			<td>Exometry</td>
			<td></td>
			<td>Organo silica beads for scatter calibration</td>
		</tr>
	</tbody>
</table>

setting a successful sorting for extracellular vesicle isolation

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. Therefore, purified MSC-EVs are envisioned as a next-generation therapeutic option for a wide array of diseases. In this protocol, we report the strategy for successfully sorting EVs from the supernatant of adipose-derived MSCs (ASCs), often used in orthopedics regenerative medicine applications.
First, we described the sample preparation, focusing on EV isolation and labeling steps with carboxyfluorescein succinimidyl ester (CFSE) for fluorescence detection; subsequently, we detailed the sorting process, which constitutes the main part of the protocol. 
In addition to the rules defined by MISEV 2023 and MIFlowCyt EV guidelines, we applied specific experimental conditions concerning nozzle size, frequency, and sheath pressure. Morphological parameters are established using beads of diameters selected to cover the theoretical range of EV size. After ASC-EVs sorting, we performed a purity check of the sorted fraction by re-analyzing it with the sorter and verifying the EV size distribution with the nanoparticle tracking analysis technique.
Due to the increasing importance of EVs, having a pure population to study and characterize is becoming crucial. Here, we demonstrate a winner strategy to set up sorting to achieve this goal.

The FSC polystyrene beads have been sorted to validate the instrument setup and sorting conditions. FSC polystyrene beads are a mix of fluorescent beads ranging from 100 nm, 300 nm, 500 nm, and 900 nm and are visible on the FITC channel. Figure 7A shows the SSC log scale versus the FITC log scale dot plot with the four populations of beads before sorting. The fluorescent populations of 100 nm, 300 nm, and 500 nm were gated and sorted. Sorted beads were analyzed for purity and enrichment as a...

Setting a Successful Sorting for Extracellular Vesicle Isolation

This protocol provides a detailed description of sorting extracellular vesicles (EVs) released by mesenchymal stromal cells. In particular, it focuses on the instrument setting and the optimization of the sorting conditions. The goal is to sort extracellular vesicles while preserving their characteristics.

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. ...

setting-a-successful-sorting-for-extracellular-vesicle-isolation

Research

JoVE Journal

Biology

759 Views.  IRCCS Ospedale San Raffaele. This protocol provides a detailed description of sorting extracellular vesicles (EVs) released by mesenchymal stromal cells. In particular, it focuses on the instrument setting and the optimization of the sorting conditions. The goal is to sort extracellular vesicles while preserving their characteristics.

Video: Setting a Successful Sorting for Extracellular Vesicle Isolation

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

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 ...

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

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 ...

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; however, there are currently no treatment or therapeutic options for vascular calcification. Calcification begins within specialized extracellular vesicles (EVs), which serve as nucleating foci by aggregating calcium and phosphate ions. This protocol describes methods for obtaining and assessing calcification in murine aortas and analyzing the associated extracted EVs. First, gross dissection of the mouse is performed to collect any relevant organs, such as the kidneys, liver, and lungs. Then, the murine aorta is isolated and excised from the aortic root to the femoral artery. Two to three aortas are then pooled and incubated in a digestive solution before undergoing ultracentrifugation to isolate the EVs of interest. Next, the mineralization potential of the EVs is determined through incubation in a high-phosphate solution and measuring the light absorbance at a wavelength of 340 nm. Finally, collagen hydrogels are used to observe the calcified mineral formation and maturation produced by the EVs in vitro.

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

This paper presents the methodology for obtaining and assessing vascular calcification by isolating murine aortas followed by extracting calcified extracellular vesicles to observe the mineralization potential.

Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; ...