TITLE: Detection and Sorting of Extracellular Vesicles and Viruses using nanoFACS
AUTHORS: Aizea Morales-Kastresana, Joshua Welsh, Jennifer Jones

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Recent advances in high resolution flow cytometry (HRFC), which show improvements in both light scatter and fluorescence sensitivity have resulted in the development of techniques that better isolate, stain and analyze single EVs(Boing et al., 2014; Groot Kormelink et al., 2016; Morales-Kastresana, Musich, Welsh, Telford, Demberg, Wood, Bigos, Ross, Kachynski, Dean, Feton, et al., 2019; Morales-Kastresana et al., 2017; Stoner et al., 2016; van der Vlist, Nolte-'t Hoen, Stoorvogel, Arkesteijn, & Wauben, 2012). Below, we describe protocols to fluorescently label EVs using CFDA-SE (hereinafter called CFSE), as well as antibodies targeted at specific EV surface proteins. We also provide guidelines for residual dye and antibody removal, appropriate data acquisition by HRFC and EV counting by HRFC. </span><span style = "font-weight:bold;">Figure 1</span><span>summarizes these protocols.</span></div><div class = "text-block"><span>The EVs used in this protocol are derived from the DC2.4 cell line, and bone marrow derived dendritic cells (BMDCs). The DC2.4 cell line are immature dendritic cells (DCs) with very low expression of typical DC markers on their surface (unpublished observation and</span><span style = "vertical-align:super;">(</span><span>Hargadon, Forrest, & Reddy, 2012)) and that release a morphologically homogeneous population of EVs (~130 nm in diameter). DC2.4 EVs will be used to demonstrate a CFSE staining in </span><span style = "font-weight:bold;">Basic Protocol 1</span><span>, as well as being used as a negative control for antibody-based staining methods (</span><span style = "font-weight:bold;">Basic Protocol 2</span><span>). Bone marrow dendritic cell (BMDC)-derived EVs are more heterogeneous in diameter (100-200 nm) and composition(Morales-Kastresana, Musich, Welsh, Telford, Demberg, Wood, Bigos, Ross, Kachynski, Dean, Felton, et al., 2019), and express DC markers such as MHC-II. BMDC EVs will be used to demonstrate antigen-specific staining with fluorochrome-conjugated antibodies in </span><span style = "font-weight:bold;">Basic Protocol 2</span><span>. DC2.4 and BMDC-derived EVs were isolated by serial ultracentrifugation, with concentration and diameter distribution characterized by NTA, as described before(Morales-Kastresana, Musich, Welsh, Telford, Demberg, Wood, Bigos, Ross, Kachynski, Dean, Feton, et al., 2019; Morales-Kastresana et al., 2017).</span></div></div>

[STEPS]
?. [Basic Protocol 1]
{"blocks":[{"key":"ekhom","text":"Carboxyfluorescein Diacetate Succynidimil Esther (CFDA-SE) is a small molecule that has been used extensively for labeling and tracking cells by flow cytometry, along with other imaging techniques(Lyons, Blake, & Doherty, 2013; Perfetto, Ambrozak, Roederer, & Koup, 2004). CFSE irreversibly binds to positively charged functional groups, such as lysine and arginine in proteins, through a covalent bond.","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"49ds8","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"e87k9","text":"The two diacetic groups in CFDA-SE\u2019s structure render the molecule highly permeable and non-fluorescent(Breeuwer et al., 1995). Therefore, it was believed that the presence of esterase, the main enzyme that catalyze the hydrolysis of the diacetic groups, is required to convert CFDA-SE to fluorescent CFSE(Wang, Duan, Liu, Fang, & Tan, 2005). However, the presence of the enzymes only accelerates the hydrolysis of CFDA-SE, due to being catalysts of this hydrolysis. Contrary to common misconceptions, esterases arenot requiredfor this hydrolysis, and other factors such as high CFSE concentration, long incubation times and high temperatures, can also promote hydrolysis of the diacetic groups(Bergsdorf, Beyer, Umansky, Werr, & Sapp, 2003; Hoefel, Grooby, Monis, Andrews, & Saint, 2003). Thus, the approach that is taken in this protocol has been optimized for staining cell culture-derived EVs with a fluorescent dye, regardless of the concentration of esterases in the EVs(Morales-Kastresana et al., 2017).","type":"unstyled","depth":0,"inlineStyleRanges":[{"length":12,"offset":515,"style":"italic"}],"entityRanges":[],"data":[]},{"key":"eb5m8","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"4j5g1","text":"The following protocol was developed to bulk stain EV populations(Thery et al., 2001; Thery, Ostrowski, & Segura, 2009). The protocol was optimized for achieving good signal to noise ratios, while avoiding excessive loss of EVs(Morales-Kastresana et al., 2017). To maximize the fluorescent intensity of the EVs, the authors recommend using CFDA-SE aliquots prepared in DMSO rather than aqueous solutions, which minimize fluorescein quenching with light and loss of diacetic groups by hydrolysis, which will render the molecule less permeant(Banks et al., 2013). This protocol can be performed in non-sterile conditions, although appropriate handling of reagents to ensure the absence of contaminating particulates is highly recommended. Although this method is very useful for purified EVs, this method is not appropriate for use in the setting of highly heterogeneous samples such as for plasma or serum, where extracellular proteins (including lipoproteins and ribonucleoproteins) not associated with EVs would be labelled along with the EV-associated proteins.","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [Basic Protocol 2]
{"blocks":[{"key":"ekhom","text":"The use of antibodies for EV staining and analysis using flow cytometry poses challenges due to the relatively large size of antibodies and fluorochrome-conjugates in comparison to the majority of EVs diameter. A conventional IgG measures ~3 x 11 nm, not including the conjugated fluorochrome(s) (Reth, 2013). Antibodies conjugated to fluorescent proteins such as PE and APC (250 and 105 kDa, respectively) are considerably bigger. Furthermore, it is known that commercial antibody preparations contain some aggregates that can be misidentified as positive events if not removed (Gagnon & Beam, 2009). Therefore, choosing a method that efficiently eliminates antibody aggregates and discriminates labeled EVs from unbound antibody conjugates is crucial. Some groups have reported successful unbound antibody removal after gradient ultracentrifugation in sucrose or iodixanol or repetitive ultracentrifugation (Groot Kormelink et al., 2016; Higginbotham et al., 2016; van der Vlist et al., 2012). In this protocol, we use high-speed centrifugation to remove antibody aggregates and size exclusion chromatography (SEC) to wash the unbound antibody after staining.","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [Basic Protocol 3]
{"blocks":[{"key":"ekhom","text":"In this protocol the authors describe the general steps to follow in order to achieve optimallaser:stream:detectors alignment and sample acquisition on the Astrios EQ flow cytometer (Beckman Coulter). The general concept however can be applied to other jet-in-air instruments.","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [Basic Protocol 4]
{"blocks":[{"key":"ekhom","text":"There are different methods to quantify the particle count in a solution. Common counting methods are resistive pulse sensing (RPS) and nanoparticle tracking analysis (NTA). RPS uses the Coulter principle to derive size from the measurable changes in electrical impedance produced by nonconductive particles suspended in an electrolyte. NTA uses tracks the movement of particles by Brownian motion allowing the calculation of their diameter using the Stokes-Einstein equation. Concentration can also be calculated by using the number of particles tracked per detection window volume. This technique requires the use of a specific instrument that may not be available to the broad scientific community on regular basis. Here, we describe an alternative method for counting particles in a solution, where spherical polystyrene fluorescent beads of known concentration are used as spike in reference materials. This method however depends upon the accuracy of the bead stock concentration.","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [Further information]
Background information, critical parameters, and troubleshooting steps can be found within the manuscript.