Journal: Physiological reports

Article Title: Circulating extracellular vesicle characteristics differ between men and women following 12 weeks of concurrent exercise training.

doi: 10.14814/phy2.16016

Figure Lengend Snippet: FIGURE 3 EV surface marker expression is altered by AHRET in men and women. The proportion of EVs expressing SGCA (a), CD9 (b), VAMP3 (c), and THSD (d) were measured via Imaging Flow Cytometry and are shown as percentage of total gated EVs. Representative images of EV particles with bright-field (BF) image of each particle and subsequent fluorescent channel images showing presence or absence of muscle-derived EVs (SGCA+), microvesicles (VAMP3+), exosomes (CD9+) and apoptotic bodies (THSD+) vesicles are shown (e). N = 9. Statistical testing was done via three-way ANOVA.

Article Snippet: Samples were then stained with the following antibodies and dilutions: anti- human CD9 Alexa Fluor 700 (1:300 dilution; Novus Biologicals, CO), anti- human VAMP3 Alexa Fluor 405 (1:300; Novus Biologicals), anti- human thrombospondin (THSD- 1) Alexa Fluor 594 (1:100; Novus Biologicals), alpha sarcoglycan (SGCA) FITC (1:400; Biorbyt, St Louis, MO).

Techniques: Marker, Expressing, Imaging, Flow Cytometry, Derivative Assay

Journal: Transfusion Medicine and Hemotherapy

Article Title: Inter-Laboratory Comparison of Extracellular Vesicle Isolation Based on Ultracentrifugation

doi: 10.1159/000508712

Figure Lengend Snippet: FACS-based characterization of isolated EVs (second round). A FACS histograms depicting the relative fluorescence/marker intensity of EV preparation 2.1 (black line) against unstained EV particle control (grey line). B Corresponding marker expression in HCT116 cells (extracellular staining for CD9, CD63, and CD81 and intracellular staining for Alix, TSG101, and calnexin). C, E Mean fluorescence intensity (MFI) raw values of CD63 (C) and CD81 (E) marker expression from laboratories 2.1–2.4. D, F MFI values per particle concentration of CD63 (D) and CD81 (F; left y axis) against the respective particle concentration per ml CCM (right y axis).

Article Snippet: HCT116-derived EVs were captured on anti-human CD9 beads for flow detection (Exosome-Human CD9 beads; Thermo Fisher Scientific).

Techniques: Isolation, Fluorescence, Marker, Control, Expressing, Staining, Concentration Assay

Journal: Scientific Reports

Article Title: Neutrophil-derived miR-223 as local biomarker of bacterial peritonitis

doi: 10.1038/s41598-019-46585-y

Figure Lengend Snippet: Stabilisation of miR-223 in PD effluent by extracellular vesicles. ( A ) miR-223 levels in cell-free effluent from infected (top, n = 5) and stable (bottom, n = 4) PD patients, before (control) and after differential centrifugation to pellet cellular debris, larger microvesicles and smaller exosomes. Data are shown as mean values ± SD, in relation to the amount of miR-223 present in the unspun effluent (control) serving as reference. ( B ) Susceptibility of extracellular miR-223 in effluent from infected PD patients to RNase A and proteinase K treatment, before (left, n = 5) and after (right, n = 4) depletion of exosomes. Each data point corresponds to an individual patient, shown as raw 40−Ct values; lines indicate means and standard deviations. Statistical analysis was performed using Kruskal-Wallis tests combined with Dunn’s multiple comparisons tests. ( C ) Fractionation of cell-free effluent from five infected PD patients by size exclusion chromatography and detection of CD9, CD15 and human serum albumin (HSA) in each fraction using plate-bound immunoassays. miR-223 was only quantified in fractions 1, 6 (marked by the dashed line), 11 and 20. Graphs depict the relative levels of each marker compared to the fraction containing the maximum amount; lines show the means and the shaded areas the 95% confidence interval.

Article Snippet: Bound material was detected with primary antibodies against CD9 (clone 209306; R&D Systems), CD15 (clone W6D3; BD Biosciences) or human serum albumin (clone 188835; R&D Systems), and developed using goat anti-mouse-biotinylated antibodies (Perkin Elmer) and europium-conjugated streptavidin (Perkin Elmer).

Techniques: Infection, Control, Centrifugation, Fractionation, Size-exclusion Chromatography, Marker

Journal: Cureus

Article Title: Extracellular Vesicle-Associated Angiopoietin-2 and Cell Migration-Inducing Protein in Lung Cancer Progression and Brain Metastases

doi: 10.7759/cureus.80200

Figure Lengend Snippet: LLC patient: (a) CD9, (b) CD63, and (c) CD81; LCM patient: (d) CD9, (e) CD63, and (f) CD81; EVs analyzed under buffer-only control (green), isotype control (blue), patient pre-operative (red), and post-operative (orange) conditions. APC: allophycocyanin; MFI: median fluorescence intensity; EV: extracellular vesicle

Article Snippet: Tetraspanin-positive extracellular vesicles identification using a bead-based flow cytometry assay Capture beads conjugated with CD9, CD63, and CD81 antibodies from the MACSPlex Exosome Kit, Human (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany, catalog no. 130-122-209) were used to bind tetraspanin-positive EVs.

Techniques: Control, Fluorescence

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: In Vivo Mobilization and Functional Characterization of Non-Human Primate Monocytic Myeloid-Derived Suppressor Cells

doi: 10.1111/ajt.13454

Figure Lengend Snippet: Flow-sorted rhesus monocytic MDSC obtained from cryopreserved leukapheresis products were added to immunobead-stimulated (anti-CD3/CD28), CFSE-labeled autologous pan T cells (1MDSC:2 T cells) at the start of 96-hr cultures. (A) Representative data showing the incidences of CD62L+CD45RA+ (naïve; TN), CD62L+CD45RA− (central memory; TCM), CD62L−CD45RA− (effector memory; TEM) and CD62L−CD45RA+ (terminally-differentiated effector memory; TEMRA) cells among gated CD4+and CD8+ T cell populations at 96 hr. (B) Overall ratios of CD4+Foxp3+CD25hi Treg:CD4+ Tn, Temra or Tmem (n=4 experiments using MDSC from 4 separate monkeys). Data are means ±1SD; †, p<0.05

Article Snippet: Abs and FACS staining procedure for flow cytometric analysis The following anti-human/rhesus monkey cross-reactive Abs (Abs) were used to examine the frequency of monocytic MDSC, CD4 + and CD8 + naïve (Tn) and memory T cells (Tmem) and CD4+ Treg by multi-color fluorescence-activated cell staining (FACS) analysis: anti-CD3 (clone # SP34–2), −CD4 (L200), −CD8 (RPA-T8), −CD11b (ICRF44), −CD14 (M5E5), −CD62L (SK11), − CD45RA (5H9), −HLA-DR (G46–6) (all BD Pharmingen, San Diego, CA), anti-CD20 (2H7), − CD274 (=B7-H1; programmed death ligand-1 [PD-L1]) (29E.2A3) (both Biolegend, San Diego, CA), anti-CD33 (AC104.3E3), −FoxP3 (PCH101) (both Miltenyi Biotec, Auburn, CA) and anti-CD25 (BC96), (eBioscience, San Diego, CA).

Techniques: Labeling