Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry.

Flow cytometry allows to characterize nanoparticles (NPs) and extracellular vesicles (EVs) but results are often expressed in arbitrary units of fluorescence. We evaluated the precision and accuracy of molecules of equivalent soluble fluorophores (MESF) beads for calibration of NPs and EVs. Firstly, two FITC-MESF bead sets, 2 and 6 um in size, were measured on three flow cytometers. We showed that arbitrary units could not be compared between instruments but after calibration, comparable FITC MESF units were achieved. However, the two calibration bead sets displayed varying slopes that were consistent across platforms. Further investigation revealed that the intrinsic uncertainty related to the MESF beads impacts the robust assignment of values to NPs and EVs based on extrapolation into the dim fluorescence range. Similar variations were found with PE MESF calibration. Therefore, the same calibration materials and numbers of calibration points should be used for reliable comparison of submicron sized particles.

Physical association of low density lipoprotein particles and extracellular vesicles unveiled by single particle analysis.

Extracellular vesicles (EVs) in blood plasma are recognized as potential biomarkers for disease. Although blood plasma is easily obtainable, analysis of EVs at the single particle level is still challenging due to the biological complexity of this body fluid. Besides EVs, plasma contains different types of lipoproteins particles (LPPs), that outnumber EVs by orders of magnitude and which partially overlap in biophysical properties such as size, density and molecular makeup. Consequently, during EV isolation LPPs are often co-isolated. Furthermore, physical EV-LPP complexes have been observed in purified EV preparations. Since co-isolation or association of LPPs can impact EV-based analysis and biomarker profiling, we investigated the presence and formation of EV-LPP complexes in biological samples by using label-free atomic force microscopy, cryo-electron tomography and synchronous Rayleigh and Raman scattering analysis of optically trapped particles and fluorescence-based high sensitivity single particle flow cytometry. Furthermore, we evaluated the impact on flow cytometric analysis in the presence of LPPs using in vitro spike-in experiments of purified tumour cell line-derived EVs in different classes of purified human LPPs. Based on orthogonal single-particle analysis techniques we demonstrate that EV-LPP complexes can form under physiological conditions. Furthermore, we show that in fluorescence-based flow cytometric EV analysis staining of LPPs, as well as EV-LPP associations, can influence quantitative and qualitative EV analysis. Lastly, we demonstrate that the colloidal matrix of the biofluid in which EVs reside impacts their buoyant density, size and/or refractive index (RI), which may have consequences for down-stream EV analysis and EV biomarker profiling.

A compendium of single extracellular vesicle flow cytometry.

Flow cytometry (FCM) offers a multiparametric technology capable of characterizing single extracellular vesicles (EVs). However, most flow cytometers are designed to detect cells, which are larger than EVs. Whereas cells exceed the background noise, signals originating from EVs partly overlap with the background noise, thereby making EVs more difficult to detect than cells. This technical mismatch together with complexity of EV-containing fluids causes limitations and challenges with conducting, interpreting and reproducing EV FCM experiments. To address and overcome these challenges, researchers from the International Society for Extracellular Vesicles (ISEV), International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH) joined forces and initiated the EV FCM working group. To improve the interpretation, reporting, and reproducibility of future EV FCM data, the EV FCM working group published an ISEV position manuscript outlining a framework of minimum information that should be reported about an FCM experiment on single EVs (MIFlowCyt-EV). However, the framework contains limited background information. Therefore, the goal of this compendium is to provide the background information necessary to design and conduct reproducible EV FCM experiments. This compendium contains background information on EVs, the interaction between light and EVs, FCM hardware, experimental design and preanalytical procedures, sample preparation, assay controls, instrument data acquisition and calibration, EV characterization, and data reporting. Although this compendium focuses on EVs, many concepts and explanations could also be applied to FCM detection of other particles within the EV size range, such as bacteria, lipoprotein particles, milk fat globules, and viruses.

Variation in haplotypes in single cysts of assemblages C and D, but not of assemblage E of Giardia duodenalis.

BACKGROUND: Giardia duodenalis, a single-celled intestinal parasite, is divided into eight assemblages (A-H), with differences in host specificity. Giardia duodenalis reproduces asexually and cycles between the binucleated trophozoite (4 N) and the infectious cyst with four nuclei (16 N). Interaction between the nuclei is limited. Therefore, genetic drift causes differences in genetic make-up between the non-daughter nuclei; the allelic sequence heterozygosity (ASH). The ASH is low (0.01%-0.0023%) for the related assemblages A and E, higher (0.43-0.53) for assemblage B and much higher (0.74% -0.89%) for the assemblage C and D at the root of the phylogenetic tree. The heterozygosity in assemblage F, in the same clade as assemblage A and E, was unknown. The heterozygosity in the sequences of the gdh and dis3 genes was used as proxy for the ASH and whole genome amplification of single cysts followed by cloning and Sanger sequencing of dis3 fragment could reveal the genetic variation within the cyst. The aim of the study was to determine the level of heterozygosity within pooled and single cysts of different assemblages. RESULTS: The heterozygosity in gdh and dis3 was determined in pooled cysts of the assemblages A to F. Heterozygosity in the isolates of the assemblages C (n = 2) and D (n = 1) ranged from 0.41% to 0.82% for gdh and dis3 and no heterozygosity was found in the isolates of the assemblages A (n = 4), E (n = 3) and F (n = 3). The heterozygosity in assemblage B (n = 7) was intermediate (0% to 0.62%). Next, the number of haplotypes of dis3 was determined for single cysts of assemblages C, D and E. In the assemblages C and D, two to four haplotypes were found per cyst, while in assemblage E only one haplotype was identified. CONCLUSIONS: Having high heterozygosity is characteristic for the assemblages C and D, while having a low heterozygosity is characteristic for the clade with the assemblages A, E and F. Presence of more than 1 haplotype per cyst in assemblage C and D suggests differences between the non-daughter nuclei, in contrast to the one haplotype in assemblage E.

Presence of Saccharomyces cerevisiae subsp. diastaticus in industry and nature and spoilage capacity of its vegetative cells and ascospores.

Saccharomyces cerevisiae sub-species diastaticus (S. diastaticus) is the main fungal cause of spoilage of carbonated fermented beverages in the brewing industry. Here, prevalence of S. diastaticus in nature and breweries was assessed as well as the spoilage capacity of its vegetative cells and spores. S. diastaticus could only be enriched from 1 out of 136 bark and soil samples from the Netherlands, being the first described natural isolate of this yeast outside South America. On the other hand, it was identified by PCR and selective enrichment in 25 and 21 out of 54 biofilm samples from beer filling halls in Asia, Africa, Europe and North America. ITS sequencing revealed that S. cerevisiae (including S. diastaticus) represented <0.05% of fungal DNA in 17 out of 20 samples, while it represented 0.1, 2 and 32% in samples VH6, VH1 and VH3 respectively. Next, vegetative cells and ascospores of the natural S. diastaticus isolate MB523 were inoculated in a variety of beer products containing 0.0-5.0% alcohol (v/v). Ascospores spoiled all beer products, while vegetative cells did not grow in Radler lemon 0.0, Radler lime mint 0.0 and Radler lemon lime 0.0. Notably, vegetative cells could spoil these Radlers when they first had been grown in alcohol free beer either or not mixed with Radler lemon lime 0.0. Conversely, vegetative cells that had been grown in Radler lemon lime lost their spoilage potential of this beer product when they had grown in YPD medium for more than 24 h. In addition, it was shown that cells grown in alcohol free beer were more heat resistant than cells grown in YPD (D52 40 min and ≤ 10.3 min, respectively). Together, these data show that S. diastaticus is a less prevalent variant of S. cerevisiae in nature, while it accumulates in breweries in mixed biofilms. Data also show that both vegetative cells and spores can spoil all tested beer products, the latter cell type irrespective of its environmental history.

Orally Administered 5-aminolevulinic Acid for Isolation and Characterization of Circulating Tumor-Derived Extracellular Vesicles in Glioblastoma Patients.

Background: In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. Extracellular vesicles (EVs) are small, cell-derived vesicles that contain miRNAs, proteins, and lipids, and possible candidates for liquid biopsies. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cells. In this study, we assessed whether PpIX accumulates in GB-derived EVs and whether these EVs could be isolated and characterized to enable a liquid biopsy in GB. Methods: EVs were isolated from the conditioned media of U87 cells treated with 5-ALA by differential ultracentrifugation. Blood samples were collected and processed from healthy controls and patients undergoing 5-ALA guided surgery for GB. High-resolution flow cytometry (hFC) enabled detection and sorting of PpIX-positive EVs, which were subsequently analyzed by digital droplet PCR (ddPCR). Results: PpIX-positive EVs could be detected in conditioned cell culture media as well as in patient samples after administration of 5-ALA. By using hFC, we could sort the PpIX-positive EVs for further analysis with ddPCR, which indicated the presence of EVs and GB-associated miRNAs. Conclusion: GB-derived EVs can be isolated from the plasma of GB patients by using 5-ALA induced fluorescence. Although many challenges remain, our findings show new possibilities for the development of blood-based liquid biopsies in GB patients.

Improved Flow Cytometric Light Scatter Detection of Submicron-Sized Particles by Reduction of Optical Background Signals.

Flow cytometry allows multiparameter analysis on a single-cell basis and is currently the method of choice to rapidly assess heterogeneity of cell populations in suspension. With the research field of extracellular vesicles (EV) rapidly expanding, there is an increased demand to address heterogeneity of EV populations in biological samples. Although flow cytometry would be the ideal technique to do so, the available instruments are in general not equipped to optimally detect the dim light scatter signals generated by submicron-sized particles like EV. Although sideward scatter light and fluorescence are currently used as a threshold signal to identify EV within samples, the forward scatter light (FSC) parameter is often neglected due to the lack of resolution to distinguish EV-related signals from noise. However, after optimization of FSC detection by adjusting the size of the obscuration bar, we recently showed that certain EV-subsets could only be identified based on FSC. This observation made us to further study the possibilities to enhance FSC-detection of submicron-sized particles. By testing differently sized obscuration bars and differently sized pinholes in the focal plane behind the FSC detection lens, we generated a matrix that allowed us to determine which combination resulted in the lowest optical background in terms of numbers of events regarding FSC detection of submicron-sized particles. We found that a combination of an 8-mm obscuration bar and a 200-µm pinhole reduced optical background in a reproducible manner to such extent that it allowed a robust separation of 100-nm polystyrene beads from background signals within the FSC channel, and even allowed thresholding on FSC without the interference of massive background signals when both beads and EV were measured. These technical adaptations thus significantly improved FSC detection of submicron-sized particles and provide an important lead for the further development and design of flow cytometers that aid in detection of submicron-sized particles. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

In vitro Chicken Bone Marrow-Derived Dendritic Cells Comprise Subsets at Different States of Maturation.

Research in chickens has been fundamental for the discovery of basic aspects of the immune system and has led to an interest in the in-depth characterization of avian immune cell types including dendritic cells (DCs). The in vitro generation and expansion of chicken bone marrow-derived DCs (chBMDCs) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) has provided a way to study chicken DCs, which are only present at limited cell numbers in vivo. This method has been employed to study the interactions between chicken DCs and pathogens or vaccines. However, a detailed characterization of the chBMDC culture is still lacking. In the present study, we performed an elaborate phenotypical and functional analysis of the chBMDC culture and addressed its heterogeneity. After 8 days of culture, chBMDCs comprised major histocompatibility complex class II (MHC-II)low and MHC-IIhigh subsets with different morphologies. Compared with MHC-IIlow chBMDCs, the MHC-IIhigh subset showed a more mature phenotype, with higher expressions of CD1.1, CD40, CD80, CCR7, and CD83, and a relatively low opsonophagocytic capacity. Nevertheless, MHC-IIhigh chBMDCs did not show an increased capacity to induce T-cell proliferation. Therefore, MHC-IIhigh chBMDCs were found to be semi-mature. Interestingly, the presence of the semi-mature MHC-IIhigh chBMDC subset reduced when cells were cultured in the presence of IL-4. Finally, prolonged cell culture after fluorescence-activated cell sorting (FACS) converted the semi-mature MHC-IIhigh subset back into the immature phenotype of the MHC-IIlow subset, demonstrating plasticity of their maturation state. This detailed characterization explained the heterogeneity of the chBMDC culture by the simultaneous presence of immature and semi-mature chBMDC subsets, in addition to cells without features of antigen-presenting cells. Our findings are instrumental for the interpretation of experiments using the chBMDC culture in past and future research by providing insights into its phenotypically and functionally distinct cell types.

MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments.

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.

Picornavirus infection induces temporal release of multiple extracellular vesicle subsets that differ in molecular composition and infectious potential.

Several naked virus species, including members of the Picornaviridae family, have recently been described to escape their host cells and spread infection via enclosure in extracellular vesicles (EV). EV are 50-300 nm sized lipid membrane-enclosed particles produced by all cells that are broadly recognized for playing regulatory roles in numerous (patho)physiological processes, including viral infection. Both pro- and antiviral functions have been ascribed to EV released by virus-infected cells. It is currently not known whether this reported functional diversity is a result of the release of multiple virus-containing and non-virus containing EV subpopulations that differ in composition and function. Using encephalomyocarditis virus infection (EMCV, Picornaviridae family), we here provide evidence that EV populations released by infected cells are highly heterogeneous. Virus was contained in two distinct EV populations that differed in physical characteristics, such as sedimentation properties, and in enrichment for proteins indicative of different EV biogenesis pathways, such as the plasma membrane resident proteins Flotillin-1 and CD9, and the autophagy regulatory protein LC3. Additional levels of EV heterogeneity were identified using high-resolution flow cytometric analysis of single EV. Importantly, we demonstrate that EV subsets released during EMCV infection varied largely in potency of transferring virus infection and in their kinetics of release from infected cells. These data support the notion that heterogeneous EV populations released by virus-infected cells can exert diverse functions at distinct time points during infection. Unraveling the compositional, temporal and functional heterogeneity of these EV populations using single EV analysis technologies, as employed in this study, is vital to understanding the role of EV in virus dissemination and antiviral host responses.

Mast Cell Degranulation Is Accompanied by the Release of a Selective Subset of Extracellular Vesicles That Contain Mast Cell-Specific Proteases.

Mast cells (MC) are well known for their effector role in allergic disorders; moreover, they are associated with diverse modulatory effects in innate and adaptive immunity. It is largely unclear how MC exert these modulating functions. In this article, we show that IgE-mediated MC degranulation leads to a rapid release of high quantities of extracellular vesicles (EV), comparable to the release of preformed mediators. EV are submicron structures composed of lipid bilayers, proteins, and nucleic acids that are released by cells in a regulated fashion and are involved in intercellular communication. Primary murine mucosal-type MC and connective tissue-type MC released phenotypically different EV populations depending on the stimulus they received. Although unstimulated MC constitutively released CD9+ EV, degranulation was accompanied by the release of CD63+ EV, which correlated with release of the soluble mediator ß-hexosaminidase. This CD63+ EV subset was smaller and exhibited a higher buoyant density and distinct phospholipid composition compared with CD9+ EV. Marked differences were observed for phosphatidylinositol, phosphatidic acid, and bis(monoacylglycero)phosphate species. Strikingly, proteomic analysis of CD63+ EV from connective tissue-type MC unveiled an abundance of MC-specific proteases. With regard to carboxypeptidase A3, it was confirmed that the enzyme was EV associated and biologically active. Our data demonstrate that, depending on their activation status, MC release distinct EV subsets that differ in composition and protease activity and are indicative of differential immunological functions. Concerning the strategic tissue distribution of MC and the presence of degranulated MC in various (allergic) disorders, MC-derived EV should be considered potentially important immune regulators.

Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles.

Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery.

Circulating Extracellular Vesicles Contain miRNAs and are Released as Early Biomarkers for Cardiac Injury.

Plasma-circulating microRNAs have been implicated as novel early biomarkers for myocardial infarction (MI) due to their high specificity for cardiac injury. For swift clinical translation of this potential biomarker, it is important to understand their temporal and spatial characteristics upon MI. Therefore, we studied the temporal release, potential source, and transportation of circulating miRNAs in different models of ischemia reperfusion (I/R) injury. We demonstrated that extracellular vesicles are released from the ischemic myocardium upon I/R injury. Moreover, we provided evidence that cardiac and muscle-specific miRNAs are transported by extracellular vesicles and are rapidly detectable in plasma. Since these vesicles are enriched for the released miRNAs and their detection precedes traditional damage markers, they hold great potential as specific early biomarkers for MI.

Identification of CD90 as Putative Cancer Stem Cell Marker and Therapeutic Target in Insulinomas.

The long-term prognosis after surgical resection of malignant insulinoma (INS) is poor. Novel adjuvant therapies, specifically targeting cancer stem cells (CSCs), are warranted. Therefore, the goal of this study was to characterize and target putative INS CSCs. Using fluorescence-activated cell sorting, human INS cell line CM and pancreatic carcinoid cell line BON1 were screened for the presence of stem cell-associated markers. CD90, CD166, and GD2 were identified as potential CSC markers. Only CD90(+) INS cells had an increased tumor-initiating potential in athymic nude mice. Anti-CD90 monoclonal antibodies decreased the viability and metastatic potential of injected cells in a zebrafish embryo INS xenograft model. Primary INS stained positive for CD90 by immunohistochemistry, however also intratumoral fibroblasts and vascular endothelium showed positive staining. The results of this study suggest that anti-CD90 monoclonals form a potential novel adjuvant therapeutic modality by targeting either INS cells directly, or by targeting the INS microenvironment.

Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry.

Submicron-sized vesicles released by cells are increasingly recognized for their role in intercellular communication and as biomarkers of disease. Methods for high-throughput, multi-parameter analysis of such extracellular vesicles (EVs) are crucial to further investigate their diversity and function. We recently developed a high-resolution flow cytometry-based method (using a modified BD Influx) for quantitative and qualitative analysis of EVs. The fact that the majority of EVs is <200 nm in size requires special attention with relation to specific conditions of the flow cytometer, as well as sample concentration and event rate. In this study, we investigated how (too) high particle concentrations affect high-resolution flow cytometry-based particle quantification and characterization. Increasing concentrations of submicron-sized particles (beads, liposomes, and EVs) were measured to identify coincidence and swarm effects, caused by the concurrent presence of multiple particles in the measuring spot. As a result, we demonstrate that analysis of highly concentrated samples resulted in an underestimation of the number of particles and an interdependent overestimation of light scattering and fluorescence signals. On the basis of this knowledge, and by varying nozzle size and sheath pressure, we developed a strategy for high-resolution flow cytometric sorting of submicron-sized particles. Using the adapted sort settings, subsets of EVs differentially labeled with two fluorescent antibodies could be sorted to high purity. Moreover, sufficient numbers of EVs could be sorted for subsequent analysis by western blotting. In conclusion, swarm effects that occur when measuring high particle concentrations severely hamper EV quantification and characterization. These effects can be easily overlooked without including proper controls (e.g., sample dilution series) or tools (e.g., oscilloscope). Providing that the event rate is well controlled, the sorting strategy we propose here indicates that high-resolution flow cytometric sorting of different EV subsets is feasible.

The paracrine feedback loop between vitamin D3 (1,25(OH)2D3) and PTHrP in prehypertrophic chondrocytes.

The endocrine feedback loop between vitamin D3(1,25(OH)2D3) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH-related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)2D3 and PTH. This was investigated in ATDC5 cells treated with 10(-8) M 1,25(OH)2D3 or PTHrP, Col2-pd2EGFP transgenic mice, and primary Col2-pd2EGFP growth plate chondrocytes isolated by FACS, using RT-qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)2D3 receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)2D3 decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α- and 24-hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)2D3 treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate. 1,25(OH)2D3 decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)2D3 by increasing VDR production. In light of the role of 1,25(OH)2D3 and PTHrP in modulating chondrocyte differentiation, 1,25(OH)2D3 in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration.

Activated peritoneal cavity B-1a cells possess regulatory B cell properties.

Previous studies have suggested that murine peritoneal cavity-derived B-1a cells possess similarities with described regulatory B cell subsets. The aim of the current study was to examine the potential immunoregulatory function of peritoneal cavity-derived B(-1a) cells. In vitro activation of peritoneal cavity-derived B- and B-1a cells shows that activation of these B cells with anti-CD40 and LPS induces these cells to secrete more IL-10, IL-6 and IgM as compared to splenic B cells. In a suppression assay, CD40/TLR4-activated peritoneal cavity B cells possess regulatory B cell functions as they inhibit the capacity of CD4(+) T cells to produce both tumor necrosis factor-α and interferon-γ. Splenic B cells did not show this, whereas non-activated peritoneal cavity B cells augmented the capacity of CD4(+) T cells to produce tumor necrosis factor-α, while the ability to produce interferon-γ was not altered. The current paper compares splenic B cells to peritoneal cavity B(-1a) cells in an in vitro activation- and an suppression-assay and concludes that peritoneal cavity B(-1a) cells possess properties that appear similar to splenic autoimmune-suppressive regulatory B cell subsets described in the literature.

Dynamics of dendritic cell-derived vesicles: high-resolution flow cytometric analysis of extracellular vesicle quantity and quality.

Nano-sized membrane vesicles are secreted by many cell types. These vesicles can serve as carriers of cellular information. DC-derived vesicles can be targeted to other immune cells and modify their function. Accurate analysis of quantitative and qualitative changes in EV production by DC upon different activation stimuli is needed to further reveal the immune regulatory properties of DC-derived EVs. However, methods for reliable quantification of individual EVs and for analysis of the heterogeneity of EV populations are limited. With our recently developed high-resolution flow cytometry-based method, we can perform a high-throughput, multiparameter, and quantitative analysis of individual EVs. With the use of this novel technique, we show that despite previous assumptions, stimulation with bacterial LPS increases EV release by DC. Furthermore, we demonstrate heterogeneity in DC-derived EVs regarding their buoyant density and MHC class II content. Finally, we show that cognate interaction between LPS-stimulated DC and CD4(+) T cells affects both the quantity and quality of LPS DC-derived EVs present in the culture supernatant. These data indicate that flow cytometry-based analysis of individual EVs is a valuable, novel tool to study the dynamics of EV secretion and composition, offering great opportunities to unveil the function of immune cell-derived EVs.

Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry.

We provide a protocol for a high-resolution flow cytometry-based method for quantitative and qualitative analysis of individual nano-sized vesicles released by cells, as developed and previously described by our group. The method involves (i) bright fluorescent labeling of cell-derived vesicles and (ii) flow cytometric analysis of these vesicles using an optimized configuration of the commercially available BD Influx flow cytometer. The method allows the detection and analysis of fluorescent cell-derived vesicles of ∼100 nm. Integrated information can be obtained regarding the light scattering, quantity, buoyant density and surface proteins of these nano-sized vesicles. This method can be applied in nanobiology to study basic aspects of cell-derived vesicles. Potential clinical applications include the detailed analysis of vesicle-based biomarkers in body fluids and quality control analysis of (biological) vesicles used as therapeutic agents. Isolation, fluorescent labeling and purification of vesicles can be done within 24 h. Flow cytometer setup, calibration and subsequent data acquisition can be done within 2-4 h by an experienced flow cytometer operator.

CD4(+) T cell activation promotes the differential release of distinct populations of nanosized vesicles.

Many cell types release nanosized vesicles derived from endosomal compartments (exosomes) or the plasma membrane. Vesicles actively released by CD4(+) T cells have immune-modulatory characteristics. Using our recently developed high-resolution flow cytometry-based method for the analysis of individual nanosized vesicles, we here investigated how T cell receptor (TCR)-triggering and co-stimulatory signals influence the quantity and characteristics of nanosized vesicles released by CD4(+) T cells. We found that the number of released nanosized vesicles within the buoyant density range characteristic for exosomes (1.10-1.19 g/ml) was increased by TCR-triggering and that additional co-stimulatory signals had a potentiating effect on vesicle release. However, the increase in the number of released vesicles varied substantially between density fractions within the 1.10-1.19 g/ml range and was highest for the vesicle populations in 1.14 and 1.17 g/ml fractions. Heterogeneity was also observed within the individual density fractions. Based on lipid bilayer fluorescent labelling intensity and light scattering, 3 distinct vesicle subpopulations were identified. One vesicle subpopulation increased significantly more upon T cell activation than the other subpopulations, and this was dependent on high levels of co-stimulation. These data show that T cells release a heterogeneous population of nanosized vesicles and indicate that T cells differentially regulate the release of distinct vesicle subpopulations depending on their activation status.