Toward the isolation of exosomes by flow cytometry

In the last two decades the Extracellular Vesicles (EVs) field has attracted a lot of attention from the scientific community, especially after the discovery that EVs can shuttle functional proteins and nucleic acids between cells. Some recent studies have shown an association between tumorigenesis and increased exosomes production. Exosomes and their influence has also been reported in the establishment of new metastatic niches. Besides that, the EV field remains confusing due to numerous and ambiguous definitions, specially caused by the huge heterogeneity of the vesicles, both in composition and function. Extracellular vesicles are divided into microvesicles which are originated from the plasma membrane and exosomes which have an endosomal origin. For now, it is technically challenging to obtain a pure exosome fraction, free from non-vesicular components, due to the fact the extracellular milieu is quite complex and can contain microvesicles or apoptotic bodies similar in size and structure to exosomes. The two most used methods, ultracentrifugation and commercial kits, don’t show a good efficiency when distinguishing the exosomes fraction specifically from the microvesicles fraction. Due to this sub-optimal efficiency demonstrated by these two methods, we have decided to use Flow Cytometry to see if we can achieve better exosome purification. We will use Fluorescence-activated cell sorting (FACS) to purify endogenous exosomes. This would be quite challenging especially due to the exosomes size and heterogeneity but on the other hand, if we have success with our approach, it would be possible to do downstream analysis in order to know their protein composition, functions and elaborate some more studies to try to find some “exosome-specific” marker. This would have a huge impact in the pharmaceutical industry, both for diagnosis and therapy.Durante as últimas duas décadas, a investigação desenvolvida sobre Vesículas extracelulares (VE), atraíu o bastante interesse por parte da comunidade científica, especialmente após ter sido descoberto que as VE podem transportar proteínas funcinais e ácidos nucleicos entre diferentes células. Estudos mais recentes mostraram uma relação entre tumorogenese e um aumento na produção de exosomas. Estes foram também associados ao estabelecimento de novas metástases. Apesar de todas estas descobertas, o domínio das VE continua significativamente confuso, nomeadamente devido às numerosas e ambíguas definições utilizadas, especialmente devido ao facto da imensa heterogeneidade entre as diversas VE, tanto a nível de composição como de função. Vesículas extracellulares estão divididas em microvesículas, que são originárias da membrana plasmática, e exosomas que têm uma oigem endossomal. No presente, é tecnicamente bastante complicado de obter uma fracção de pura exosomas que não apresente componentes não vesiculares, principalmente pelo facto do meio extracellular ser bastante complexo e poder conter microvesícula e corpos apoptóticos semelhantes em termos de tamanho e estrutura. Os dois métodos mais usados, a ultracentrifugação e kits comerciais, não apresentam uma boa eficiência na distinção de exosomas, especialmente das microvesículas. Devido a esta eficiência sub-óptima demonstrada por estes dois métodos, decídimos usar a separação celular por citometria de fluxo (FACS) para proceder ao isolamento de exosomas endógenos. Este objectivo será bastante desafiador especialmente pelo tamanho e heterogeneidade dos exosomas mas, por outro lado, se formos suficientemente bem sucedidos na nossa abordagem, será possível realizar análises posteriores, de modo a conhecer a sua composição proteica, funções e partir para novos estudos de modo a tentar identificar um marcador molecular específico para exosomas. Isto teria um impacto significativo na indústria farmacêutica, tanto a nível de diagnóstico como terapêutico

Microplate-based high throughput screening procedure for the isolation of lipid-rich marine microalgae

We describe a new selection method based on BODIPY (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) staining, fluorescence activated cell sorting (FACS) and microplate-based isolation of lipid-rich microalgae from an environmental sample. Our results show that direct sorting onto solid medium upon FACS can save about 3 weeks during the scale-up process as compared with the growth of the same cultures in liquid medium. This approach enabled us to isolate a biodiverse collection of several axenic and unialgal cultures of different phyla

Detection of diaphragmatic fatigue in man by phrenic stimulation.

Transdiaphragmatic pressure (Pdi) was measured at functional residual capacity (FRC) in four normal seated subjects during supramaximal, supraclavicular transcutaneous stimulation of one phrenic nerve (10, 20, 50, and 100 Hz--0.1 ms duration) before and after diaphragmatic fatigue, produced by breathing through a high alinear inspiratory resistance. Constancy of chest wall configuration was achieved by placing a cast around the abdomen and the lower one-fourth of the rib cage. Pdi increased with frequency of stimulation, so that at 10, 20, and 50 Hz, the Pdi generated was 32 +/- 4 (SE), 70 +/- 3, and 98 +/- 2% of Pdi at 100 Hz, respectively. After diaphragmatic fatigue, Pdi was less than control at all frequencies of stimulation. Recovery for high stimulation frequencies was complete at 10 min, but at low stimulation frequencies recovery was slow: after 30 min of recovery, Pdi at 20 Hz was 31 +/- 7% of the control value. It is concluded that diaphragmatic fatigue can be detected in man by transcutaneous stimulation of the phrenic nerve and that diaphragmatic strength after fatigue recovers faster at high than at low frequencies of stimulation. Furthermore, it is suggested that this long-lasting element of fatigue might occur in patients with chronic obstructive lung disease, predisposing them to respiratory failure.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Use of differential charging to analyse microscopic specimens of Azolla leaves by XPS

Cavity cuticles that surround the bacterial symbionts residing in the leaf cavities of the aquatic fern Azolla and Azolla leaf cuticle on the epidermis outside the cavity (outer cuticle) were submitted to XPS analysis. Differential charging enabled the signals of the microscopic sample and the support signals to be differentiated. The cavity cuticle has a composition similar to cutin. In comparison with the outer cuticle, it is characterized by a lower concentration of aliphatic chains and a higher concentration of oxygen-containing functions. This has implications in term of solute transport between the leaf and the bacteria. Copyright (C) 2002 John Wiley Sons, Ltd

Protein-energy malnutrition and anemia in Kivu

info:eu-repo/semantics/publishe

Guidelines for establishing a cytometry laboratory

The purpose of this document is to provide guidance for establishing and maintaining growth and development of flow cytometry shared resource laboratories. While the best practices offered in this manuscript are not intended to be universal or exhaustive, they do outline key goals that should be prioritized to achieve operational excellence and meet the needs of the scientific community. Additionally, this document provides information on available technologies and software relevant to shared resource laboratories. This manuscript builds on the work of Barsky et al. 2016 published in Cytometry Part A and incorporates recent advancements in cytometric technology. A flow cytometer is a specialized piece of technology that require special care and consideration in its housing and operations. As with any scientific equipment, a thorough evaluation of the location, space requirements, auxiliary resources, and support is crucial for successful operation. This comprehensive resource has been written by past and present members of the International Society for Advancement of Cytometry (ISAC) Shared Resource Laboratory (SRL) Emerging Leaders Program https:// isac-net.org/general/custom.asp?page=SRL-Emerging-Leaders with extensive expertise in managing flow cytometry SRLs from around the world in different settings including academia and industry. It is intended to assist in establishing a new flow cytometry SRL, re-purposing an existing space into such a facility, or adding a flow cytometer to an individual lab in academia or industry. This resource reviews the available cytometry technologies, the operational requirements, and best practices in SRL staffing and management