Murine models in particular display a smaller degree of variability than clinical samples, which facilitates the discovery of biomarker candidates. To contour some of these limitations, pre-clinical models are frequently employed, as they permit a higher degree of control over the experimental setup and sampling, and facilitate basic research related to EV composition, biogenesis and biological functions. However, high heterogeneity, which include differences in both quantity and quality of EVs, usually represent a challenge to the identification of specific EVs biomarkers in human samples. They serve as non-invasive sources of liquid biopsies for clinical biomarkers and allow longitudinal sampling to follow disease progression. As shown by us ( Peinado et al., 2012 Costa-Silva et al., 2015 Hoshino et al., 2015 Rodrigues et al., 2019) and others ( Kalluri, 2016), EVs can act as messengers and carriers of biomolecules in physiological and pathological scenarios ( Maia et al., 2018 Stahl and Raposo, 2019).Įxtracellular vesicles have been described in all body fluids ( Kalluri and LeBleu, 2020), including blood ( Caby et al., 2005), urine ( Pisitkun et al., 2004), saliva ( Ogawa et al., 2011), and vitreous humor ( Ragusa et al., 2015). Their cargo includes proteins, lipids and nucleic acids, which can be transported both locally and to distant cellular targets through the peripheral circulation. In addition to differences in size and biogenesis mechanism, EVs are highly heterogeneous in molecular composition and biological properties ( Groot Kormelink et al., 2016 Kowal et al., 2016 Willms et al., 2016, 2018). Our FC strategy has an unexplored clinical potential to analyze EVs in biofluids with intrinsically limited volumes and to multiply the number of different analytes in EVs that can be studied from a single collection of biofluid.Įxtracellular vesicles (EVs), membrane vesicles released by all cells, are emerging players in cell-cell communication. By quantifying the proportion of vesicular particles in purified and non-purified biological samples, this method also serves as a precious tool to quality control isolates of EVs purified by different protocols. To illustrate its application, we performed longitudinal non-lethal population analysis of EVs in mouse plasma and in single-animal collections of murine vitreous humor. By doing so, it enables population analysis of EVs in samples characterized by challenging small volumes, while reducing overall sample processing time. We here present a Flow Cytometry (FC) strategy that does not require isolation or concentration of EVs prior to staining. EVs-based liquid biopsies usually require EVs isolation before content analysis, which frequently increases sample volume requirements. By carrying biomolecules from tissues to biofluids, EVs have attracted attention as non-invasive sources of clinical biomarkers in liquid biopsies. 3Digestive Unit, Champalimaud Clinical Centre, Lisbon, PortugalĮxtracellular Vesicles (EVs), membrane vesicles released by all cells, are emerging mediators of cell-cell communication.2Graduate Program in Areas of Basic and Applied Biology, University of Porto, Porto, Portugal.1Champalimaud Research, Champalimaud Centre for the Unknown, Lisbon, Portugal.Gregório 1, Cristian Bodo 1, Julia Elzanowska 1, Maria Carolina Strano Moraes 1*† and Bruno Costa-Silva 1*† It is not usually possible to do intracellular staining before sorting of live cells, as the permeabilization requires damage to the cell membrane which would compromise the cell viability.Joana Maia 1,2, Silvia Batista 1, Nuno Couto 1,3, Ana C.The experiment should be undertaken in aseptic sterile conditions to ensure the cells do not become contaminated.Avoid sodium azide in the buffers during staining as this can be toxic to cells and compromise viability.The cells need to remain viable and without contamination for subsequent culture. Sorted cell populations are then analyzed to ensure successful cell sorting.Collection tubes to the left would collect all the negatively charged PE stained cell droplets. Collection tubes to the right would collect all the positively charged FITC stained cell droplets.Ī single PE stained cell in a single droplet would be given a negative charge and be attracted to the left. Deflection plates attract or repel the cells accordingly into collection tubes.Ī single FITC stained cell in a single droplet would be given a positive charge and be attracted to the right.

This drop is given an electronic charge, depending on the fluorescence of the cell inside the drop. The machine is set up so that each individual cell then enters a single droplet as it leaves the nozzle tip.Individual cells are 'interrogated' by the laser as in a normal flow cytometer.

Facs analysis