Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV-FC researchers from ISEV, ISAC and ISTH, worked together as an EV-FC WG and developed a consensus framework for the minimum information that should be provided regarding EV-FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV-FC-specific reporting framework (MIFlowCyt-EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV-FC data. MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt-EV will ensure consistency in the manner of reporting of EV-FC studies, over time we expect that adoption of MIFlowCyt-EV as a standard for reporting EV-FC studies will improve the ability to quantitatively compare results from different laboratories and to support the development of new instruments and assays for improved measurement of EVs.

Extracellular vesicles (EVs) are attracting attention as vehicles for inter-cellular signaling that may have value as diagnostic or therapeutic targets. EVs are released by many cell types and by different mechanisms, resulting in phenotypic heterogeneity that makes them a challenge to study. Flow cytometry is a popular tool for characterizing heterogeneous mixtures of particles such as cell types within blood, but the small size of EVs makes them difficult to measure using conventional flow cytometry. To address this limitation, a high sensitivity flow cytometer was constructed and EV measurement approaches that allowed them to enumerate and estimate the size of individual EVs, as well as measure the presence of surface markers to identify phenotypic subsets of EVs. Several fluorescent membrane probes were evaluated and it was found that the voltage sensing dye di-8-ANEPPS could produce vesicle fluorescence in proportion to vesicle surface area, allowing for accurate measurements of EV number and size. Fluorescence-labeled annexin V and anti-CD61 antibody was used to measure the abundance of these surface markers on EVs in rat plasma. It was shown that treatment of platelet rich plasma with calcium ionophore resulted in an increase in the fraction of annexin V and CD61-positive EVs. Vesicle flow cytometry using fluorescence-based detection of EVs has the potential to realize the potential of cell-derived membrane vesicles as functional biomarkers for a variety of applications. V C 2015 International Society for Advancement of Cytometry

To the editor:Obtaining diffraction-quality crystals is a major bottleneck in protein X-ray crystallography. For example, the current success rate for protein structure solution at the Midwest Center for Structural Genomics (starting from purified protein) is ~10%. Protein crystallization is influenced by many factors, and many methods have been developed to enhance crystallization. In particular, reductive methylation of proteins has been successfully applied to obtain high-quality crystals 1-4 . Several studies 3,5,6 have indicated that methylating the solvent-exposed ε-amino group of lysines changes protein properties (pI, solubility and hydropathy) 7,8 , which may promote crystallization via improving crystal packing. Reductive methylation of proteins is a simple, generic method; it is fast, specific and requires few steps under relatively mild buffer and chemical conditions and can be executed for several proteins in parallel. Native and methylated proteins have very similar structures, and, in most cases, methylated proteins maintain their biochemical function 2,5,9 . Some proteins can only be crystallized after methylation 3,10 , and crystals of modified proteins often diffract to higher resolution 3,9 . The efficacy of the method has been previously tested on 10 proteins, with a 30% success rate 3 .Here we investigated the application of reductive methylation on a large scale. We applied a previously described reductive methylation protocol 2,11 (Supplementary Methods online) to 370 sequence-diverse proteins selected from protein families that had no structural homologs with >30% sequence identity. We expressed 370 recombinant proteins and purified them using standard methods 12 and screened them using standard crystal screening methods (Supplementary Methods). Of the 370 proteins, 269 proteins had not previously yielded crystals suitable for structure determination (crystals were too small, poorly ordered, twinned, highly mosaic or multiple), 85 proteins had previously failed to crystallize and 16 proteins were a reference set (not previously screened for crystallization; Table 1 and Supplementary Tables 1 and 2 online). After reductive methylation, we obtained diffraction-quality crystals for 40 of the 370 proteins, and so far we solved 26 crystal structures ( NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptWe also determined the structures of 4 proteins in their native as well as their methylated states (Supplementary Methods). By comparing these structures, we obtained insight into how methylation affects protein crystallization. We observed a decrease in the isotropic B factor (Fig. 1), which is likely a result of more ordered crystal packing and which leads to better diffraction limits. Indeed, the resolution of the methylated structures (average, 2.07 Å) was better than that of their native counterparts (average, 3.05 Å; Supplementary Table 1). The methylated lysines were engaged in various intra-and intermolecular interactions with protein and solvent (carboxylates and ...

Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patients. However, the optimal method for quantitative assessment of EVs in clinical bio-fluid remains a point of contention. Multiple high-resolution platforms for quantitative EV analysis have emerged, including methods grounded in diffraction measurement of Brownian motion (NTA), tunable resistive pulse sensing (TRPS), vesicle flow cytometry (VFC), and transmission electron microscopy (TEM). Here we compared quantitative EV assessment using cerebrospinal fluids derived from glioblastoma patients using these methods. For EVs <150 nm in diameter, NTA detected more EVs than TRPS in three of the four samples tested. VFC particle counts are consistently 2–3 fold lower than NTA and TRPS, suggesting contribution of protein aggregates or other non-lipid particles to particle count by these platforms. While TEM yield meaningful data in terms of the morphology, its particle count are consistently two orders of magnitude lower relative to counts generated by NTA and TRPS. For larger particles (>150 nm in diameter), NTA consistently detected lower number of EVs relative to TRPS. These results unveil the strength and pitfalls of each quantitative method alone for assessing EVs derived from clinical cerebrospinal fluids and suggest that thoughtful synthesis of multi-platform quantitation will be required to guide meaningful clinical investigations.