Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80–200 nm, microvesicles: ~200–1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for routine quantification and characterization of EVs from various sources. Finally, it has the potential to bring a desired level of control into routine experiments and non-specialized labs, thanks to its simple bead-based standardization.

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The five highly related envelope subgroups of the avian sarcoma and leukosis viruses (ASLVs), subgroup A [ASLV(A)] to ASLV(E), are thought to have evolved from an ancestral envelope glycoprotein yet utilize different cellular proteins as receptors. Alleles encoding the subgroup A ASLV receptors (Tva), members of the low-density lipoprotein receptor family, and the subgroup B, D, and E ASLV receptors (Tvb), members of the tumor necrosis factor receptor family, have been identified and cloned. However, alleles encoding the subgroup C ASLV receptors (Tvc) have not been cloned. Previously, we established a genetic linkage between tvc and several other nearby genetic markers on chicken chromosome 28, including tva. In this study, we used this information to clone the tvc gene and identify the Tvc receptor. A bacterial artificial chromosome containing a portion of chicken chromosome 28 that conferred susceptibility to ASLV(C) infection was identified. The tvc gene was identified on this genomic DNA fragment and encodes a 488-amino-acid protein most closely related to mammalian butyrophilins, members of the immunoglobulin protein family. We subsequently cloned cDNAs encoding Tvc that confer susceptibility to infection by subgroup C viruses in chicken cells resistant to ASLV(C) infection and in mammalian cells that do not normally express functional ASLV receptors. In addition, normally susceptible chicken DT40 cells were resistant to ASLV(C) infection after both tvc alleles were disrupted by homologous recombination. Tvc binds the ASLV(C) envelope glycoproteins with low-nanomolar affinity, an affinity similar to that of binding of Tva and Tvb with their respective envelope glycoproteins. We have also identified a mutation in the tvc gene in line L15 chickens that explains why this line is resistant to ASLV(C) infection.Retroviruses require an interaction between the viral glycoproteins and a specific cell surface protein (receptor) to initiate entry into a cell (reviewed in references 32 and 56). The envelope glycoproteins of retroviruses are composed of trimers of two glycoproteins: the surface glycoprotein (SU), which contains the domains responsible for interaction with the host receptor, and the transmembrane glycoprotein (TM), which anchors SU to the membrane and mediates fusion of the viral and host membranes. The interaction of the SU glycoprotein with the host receptor usually involves multiple, noncontiguous determinants in both proteins that specify receptor choice and binding affinity and trigger a conformational change in the envelope glycoproteins that initiates the fusion process. Despite the complexity and specificity of the interaction between the viral glycoproteins and host receptors, closely related retroviruses carry envelope glycoproteins with mutations that alter receptor usage. The natural selection of retroviral subgroups with altered receptor usage may help the virus overcome host resistance and promote coinfection and may lead to heterotransmission.The five highly related envelope subgroups of...

Retroviruses share a common strategy for entry into cells (reviewed in references 27 and 51). The entry process is initiated by an interaction between the viral envelope glycoprotein and a specific cell surface protein that acts as a receptor. This interaction triggers a conformational change in the structure of the viral glycoprotein that leads to the fusion of the viral and cellular membranes. Despite the complexity of the interaction between the viral glycoprotein and the receptor, closely related retroviruses carry envelope glycoproteins that use different cellular proteins as receptors. In the avian sarcoma and leukosis viruses (ASLVs), there are five highly related envelope subgroups, subgroups A to E, that are thought to have evolved from a common ancestor (reviewed in references 7 and 50). The presence of viral subgroups that utilize distinct receptors helps the virus overcome host resistance and promotes coinfection. We and others have developed strategies that mimic resistance to ASLV entry in cell culture to study the evolution of the envelope glycoprotein. These studies demonstrated that blocking virus entry could select viral variants with mutations in the viral glycoproteins that altered receptor usage (24,25,31,34,43). The goal of the present study was to identify and characterize the mutations in the subgroup A receptor in lines of chickens that cause resistance to infection by ASLVs carrying the subgroup A envelope glycoproteins.Three genetic loci in chicken cells determine the susceptibility and resistance to subgroup A to E ASLVs: tva (susceptibility to subgroup A viruses), tvb (susceptibility to subgroup B, D, and E viruses), and tvc (susceptibility to subgroup C viruses) (49, 50). Alleles that confer susceptibility to ASLV infection are dominant: two recessive resistance alleles are required at these loci to confer resistance. Because the tva r , tvb r , and tvc r resistance alleles are recessive, it is unlikely that these alleles encode dominant-negative forms of the receptor protein. The resistance alleles are likely to contain defects that either block receptor expression or prevent its use as an efficient ASLV receptor (29).Several alleles of the tvb genetic locus and three related Tvb receptors have been identified. Two different susceptibility alleles have been defined at the chicken tvb locus. The tvb s1 allele confers susceptibility to subgroups B, D, and E; the tvb s3 allele confers susceptibility to only subgroups B and D (1, 3). These alleles encode the chicken Tvb S1 (3) and Tvb S3 (12) receptors, respectively. Tvb S3 differs from Tvb S1 by a single amino acid change, cysteine to serine at position 62, which presumably alters the structure of the Tvb S1 protein so that it no longer functions as an ASLV(E) receptor. A third tvb receptor, the turkey Tvb T receptor (2), which confers susceptibility to only subgroup E ASLV, has also been cloned. The Tvb proteins are members of the tumor necrosis factor receptor (TNFR) family. The recessive tvb r resistance allele does not support the...