A filtration-based protocol to isolate human Plasma Membrane-derived Vesicles and exosomes from blood plasma

Grant, R.; Ansa-Addo, Ephraim; Stratton, Dan; Antwi‐Baffour, Samuel; Jorfi, Samireh; Kholia, Sharad; Krige, Lizelle; Lange, Sigrun; Inal, Jameel M.

doi:10.1016/j.jim.2011.06.024

Cited by 123 publications

(112 citation statements)

References 27 publications

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“…We therefore analyzed two transmembranes (CD63 and HLA), one intracellular (Ago-2) and two extracellular (gelatinases and plasminogen activators) proteins. CD63, particularly, was long considered a specific EXOs marker, but not more nowadays (35)(36)(37). CD63 and HLA expression is more evident in EVs obtained after 18 h of stimulation as compared to Ago-2, which seems to be slightly more expressed in EVs obtained after 30 min.…”

Section: Discussionmentioning

confidence: 99%

Time-dependent release of extracellular vesicle subpopulations in tumor CABA I cells

et al. 2015

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Abstract. Investigations into extracellular vesicles (EVs) have significantly increased since their role in physiological and pathological processes has become more clearly understood. Furthermore, it has become increasingly clear that several subpopulations of EVs exist, such as exosomes (EXOs) and microvesicles (MVs). Various methods and techniques used to identify and isolate the specific EVs subpopulations exist. However, these methods should be further elucidated. A deep understanding of the different factors that affect the EVs release may therefore be useful for the standardization of protocols and to establish guidelines for a more adequate analysis and correct inter-laboratory comparison. In the present study, we investigated whether composition and molecular features of EVs altered over time following a trigger stimulus. Starved CABA I cells were stimulated with FBS and conditioned medium was collected after different time intervals (30 min and 4, 8 and 18 h). The dynamic of EVs release was time-dependent, as shown by the results of scanning electron microscopy. Additionally, the time elapsed from the stimulus affected the size distribution (as highlighted by transmission electron microscopy and NanoSight assay), amount (in terms of the number of particles and protein amount) and molecular composition (CD63, HLA, Ago-2, gelatinases, and plasminogen activators) suggesting that, different EVs subpopulations were released at different time intervals following cell stimulation. Collectively, the results suggested that, parameters useful to standardize procedures for EVs isolation, including stimulation time should be considered.

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Section: Discussionmentioning

confidence: 99%

Time-dependent release of extracellular vesicle subpopulations in tumor CABA I cells

et al. 2015

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“…For instance, other cardiovascular risks such as smoking should be considered as factors able to modify the level and the type of circulating microparticles. Whereas Grant and collaborators [74] have described reduced levels of circulating plasma-derived microvesicles (mix of microparticles and exosomes) in smokers, other workers have found an increase in endothelial-derived microparticles [75] and unchanged levels of procoagulant microparticles [76]. Moreover, in vitro exposure of macrophages to tobacco smoke can directly induce the release of a mix of microparticles and exosomes carrying gelatinolytic and collagenolytic activities, mainly through the protease MMP14.…”

Section: Metabolic Syndrome (Mets)mentioning

confidence: 99%

Microparticles as Biomarkers of Vascular Dysfunction in Metabolic Syndrome and its Individual Components

Agouni¹,

Andriantsitohaina²,

Martinez

2014

CVP

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“…Differential centrifugation-protocol results in isolation of PMP that in many cases contain exosomes. To avoid the exosomes contamination Grant et al proposed filtration of PPP through a 0.1 μm pore size filter [Grant et al, 2011]. MP may be also isolated from PPP by capturing them using immobilized annexin V which binds to PS present on PMP and other MP.…”

Section: Methods Of Pmp Generation and Their Phenotype Analysis By Flmentioning

confidence: 99%

“…Thus, if platelet-derived MP (PMP), whose presence is predominant in plasma samples, is a subject of the study then the pellet obtained after this centrifugation step will mainly consist of them. At this step, one has to also consider the impact of size with regards to MP because if exosomes (part of MP population), which are considered to be smaller vesicles, are the subject of the study then they will remain in the supernatant, and the next centrifugation step/steps is/are considered to be crucial to obtain them (centrifugal force up to 150,000xg) [Grant et al, 2011, Al-Nedawi et al, 2008. The adopted form of verifying the individual steps of the different centrifugation protocols employs flow cytometry.…”

Section: Isolation Of Mp From Biological Fluids or Culture Supernatanmentioning

confidence: 99%

Application of Flow Cytometry in the Studies of Microparticles

Baj‐Krzyworzeka¹,

Baran²,

Szatanek³

et al. 2012

Flow Cytometry - Recent Perspectives

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A filtration-based protocol to isolate human Plasma Membrane-derived Vesicles and exosomes from blood plasma

Cited by 123 publications

References 27 publications

Time-dependent release of extracellular vesicle subpopulations in tumor CABA I cells

Time-dependent release of extracellular vesicle subpopulations in tumor CABA I cells

Microparticles as Biomarkers of Vascular Dysfunction in Metabolic Syndrome and its Individual Components

Application of Flow Cytometry in the Studies of Microparticles

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