Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Developmental shift in the apostat: Comparison of neurones and astrocytes

AbstractThe intrinsic pathway of apoptosis was investigated in cell-free extracts of neurones and astrocytes at various stages of maturation. Neuronal extracts were activated 65-fold after 3 days, 9-fold after 7 days, and were not activated after 10 days in culture. In contrast, astrocyte extracts were activated to a similar extent at all stages, up to 60 days in culture. The co-incubation of neuronal and astrocyte extracts followed by addition of cytochrome c/2′-deoxyadenosine 5′-triphosphate led to a 40-fold activation, suggesting that the development-associated neuronal shift does not involve the appearance of a dominant inhibitor, but rather downregulation of some key component(s) involved in caspase activation

Glucosamine (GlcN) Triggers Nuclear Translocation of Phosphorylated ERK.

<p>(A) Representative images of phospho-ERK (green) expression in NK-92 cells treated with either GlcN and IL2 or IL2 alone. DAPI was used to stain the NK-92 cell nuclei. The merged image shows the overlapping DAPI and phospho-ERK signals. (B) Ratios of the nuclear and cytoplasmic concentrations of p-ERK in the GlcN-treated and untreated NK-92 cells. Sixty individual cells were analyzed in three independent microscopic slides. (C) Imunoblot shows the amount of p-ERK in the nuclei and cytoplasm of control and IL-2 activated (for 60 minutes) cells, treated with GlcN or left untreated. Histone H3 and GAPDH are showing that nuclear and cytosolic fractions were clear.</p

Glucosamine (GlcN) Decreases Phosphorylation of FOXO1 and Paxillin in NK-92 Cells.

<p>(A) Representative immunoblots show the immunoprecipitation of whole-cell lysates, using anti-FOXO1 antibodies, followed by immunoblotting with anti-phospho-Ser antibodies (upper), and reblotting with anti-<i>O</i>-GlcNAc (middle) antibodies, of the NK-92 cells untreated or treated with GlcN. (B) Whole-cell lysates were subjected to immunoprecipitation with anti-paxillin antibodies. Immunoprecipitates were analyzed by immunoblotting with anti-phospho-Thr antibodies (upper), and anti- <i>O</i>-GlcNAc (middle). Total paxillin levels were analyzed using the anti-paxillin antibodies (lower).</p

Glucosamine (GlcN) Treatment Affects Cathepsin C and E Intracellular Levels and Localization in NK-92 cells.

<p>(A) Activities of specific cysteine cathepsins (substrate, Z-Phe-Arg-AMC), cathepsin E (KYS-1), and cathepsin C (H-Gly-Phe-AMC) determined in NK-92 cells treated with or without GlcN. Enzyme activities are presented as fold changes relative to the activity of these enzymes in the untreated cells. (B) Secreted cathepsin C activity following GlcN treatment. Results are presented as fold changes relative to the values obtained for the controls samples. Error bars represent standard deviations of the results obtained from five independent experiments. (C) Cathepsin E and C levels in the lysate of the untreated and GlcN-treated NK-92 cells. β-actin levels were used for normalization. (D) Cathepsin C <i>N</i>-glycosylation type in the GlcN-treated and untreated cells determined using EndoH (EH) and PNGaseF (P). (E) Subcellular localization of cathepsins C (white) and E (green) in NK-92 cells cultured with K562 cells, untreated or treated with GlcN. Cell nuclei were stained with DAPI, the right panel represent bright- filter (BF). Merged images show the overlapping signals. (F) Cathepsin C (green) and perforin (red) colocalization in GlcN-treated and untreated cells. The colocalized area panel shows colocalized area calculated by the LAS AF software.</p

Glucosamine (GlcN) Suppresses the Cytotoxic Activity of NK-92 Cells Against K562 Cells.

<p>(A) Cytotoxicity of NK-92 cells against K562 cells, following treatment of the cultures overnight (ON) with different concentrations of GlcN or <i>N</i>-acetyl GlcN (GlcNAc). (B) Cytotoxicity of NK-92 cells against K562 cells after ON pretreatment with GlcN, followed by 5-mM GlcN supplementation during the assay. All experiments were performed independently five times. Error bars represent standard deviations.</p

Glucosamine (GlcN) Prolongs ERK Phosphorylation in NK-92 cells.

<p>(A) Representative western blots showing the expression of phosphorylated P38, JNK, and ERK, and total MAPK protein levels in NK-92 cells treated with IL2 alone or in combination with GlcN. (B) Quantification of phosphorylated P38, JNK, and ERK levels in cells treated with IL2 alone or in combination with GlcN, normalized to the total protein levels. Error bars represent standard deviations.</p

Glucosamine (GlcN) Prevents Lytic Granule Polarization in NK Cells.

<p>(A) Polarization of granules expressing perforin (green) in the NK-92 and K562 cell conjugates, in presence or absence of GlcN (upper panels). Representative images of at least 60 NK-92/K562 conjugates are shown. Polarization of granules expressing perforin (green) in the mouse primary NK cell and 4T1 cell conjugates, in presence or absence of GlcN (lower panels). Representative images of at least 20 NK/4T1 conjugates are presented. Conjugates were considered polarized when the perforin signal was located in the quarter of the NK cell nearest to the target cell (merged image). Right, bright-filter images, for detection of the conjugates. (B) Distribution analysis, showing the percentage of NK-92 and K562 conjugates with polarized lytic granules in the untreated and GlcN-treated cells. (C) Western blots of NK-92 cell lysates untreated and treated with GlcN, showing the amount of perforin.</p