MOESM3 of Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions

Additional file 3: Figure S3. Antibody differential binding to cell-derived EVs. EVs derived from HEK-293, SKBR3, and Jurkat cells were used to evaluate the binding preferences of the nanobodies H1 and B1 compared with the binding of the irrelevant clone nbVHH (A–C). The binding capacity of a commercial anti-CD9 antibody was tested with the same cell lines (D). Bars indicate median percentage of positively stained EV coated beads with anti-CD9–PE antibodies to H1-GFP, B1-GFP, and non-binding VHH coated beads with respect to autofluorescence of unstained EV coated beads. The error bars indicate standard deviations for triplicate measurements

MOESM4 of Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions

Additional file 4: Figure S4. Anti-exosome nanobodies bind EV-fractions separated by chromatography. Flow cytometry experiments show that both H1 and H6 strongly bind to exosomes present in the fraction 1 separated by IEX chromatography

MOESM1 of Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions

Additional file 1: Figure S1. Chromatographic separation of kit-purified EV-enriched fraction from culture media. Kit-precipitated EVs present in SKBR3 cell culture supernatant were separated using a large-pore anion-exchange monolith column. All the four separated fractions were analyzed by flow-cytometry and resulted positive for the EV marker CD9

GSC-derived exosomes inhibit T-cell proliferation and expression of activation markers and modulate cytokine production of PBMCs.

<p>CFSE-labeled PBMCs isolated from healthy donors were pretreated for 24 hours without (white column, CTRL) or with GSC-derived exosomes (black column, GSC-EXO) and stimulated for 4 days with anti-CD3 and anti-CD28. (A) Representative microscope images and respective cytometry CFSE histograms, showing the fraction of proliferative CD3+ T cells, in unstimulated PBMCs (i), stimulated PBMCs (ii) and exosomes-treated stimulated PBMCs (iii). (B-D) Histograms showing, within the PBMCs, the fraction of proliferating CD3+ (B), CD4+ (C) and CD8+ (D) T cells. (E-F) CD3+ T-cell expression of CD25 and CD69 was measured by flow cytometry on day 2. (G-L) PBMC-derived supernatants were harvested after 48 hours and used for ELISA with the Bio-plex cytokine assay system. Cytokines that showed statistically significant differences with the exosome treatment are reported. Concentration of IL-2 (G), INF-γ (H), TNF-α (I) and IL-5 (J) are expressed as pg/ml. In B-L, the data are presented as mean ± SD (n = 6). *, <i>p</i><0.05 versus control.</p

Cytokine production by stimulated PBMCs treated with GSC-derived exosomes.

<p>Cytokine production by stimulated PBMCs treated with GSC-derived exosomes.</p

Characterization of exosomes-enriched preparation obtained from GSC.

<p>The NTA was performed on GSC-derived exosomes samples in order to quantify particle concentration normalized for the number of producing cells or millilitre of supernatants. (A) A representative graph of NTA is shown. (B) The data show the amount of exosomes produced by different GSC samples considering either the number of cells counted at the end of the 48 hours culture or the volume of cell supernatants. The data are presented as mean ± SD; n = 7. (C) Immunoblotting of the Jurkat whole cell lysate (positive control), GSC, GSC-derived ExoQuick pellet and supernatant for exosomal surface protein TSG101 (Molecular Weight, 43kDa). (D) Representative FACS histograms of CD9, CD81 and CD63 exosome-specific markers are shown.</p

GSC-derived exosomes stimulate CD25 expression and proliferation of isolated CD4+ T cells but do not affect differentiation and suppressive activity of Treg cells.

<p>CD4+ T cells, isolated from PBMCs by negative selection, were stimulated with anti-CD3, anti-CD28 and IL-2 in the absence (white column, CTRL) or presence (black column, GSC-EXO) of GSC-derived exosomes. Expression of CD25 (A), percentage of proliferative CFSE-labelled cells in the presence of indicated stimuli (B) and frequency of CD4+/CD25+/FoxP3+ (C) was determined by flow cytometry on day 4. Columns, mean (n = 6); bars, SD; *, significantly different from the control; <i>p</i><0.05. (D) CFSE-labelled purified CD4+ T cells, stimulated with anti-CD3, were co-cultured for 4 days with mitomycin-treated PBMCs and CD4+/CD25+/CD127^dim T-reg cells pre-incubated without (white column, CTRL) or with (black column, GSC-EXO) GSC-derived exosomes. Percentage of proliferative CFSE-labelled CD4+ T cells was measured by flow cytometry. Columns, mean (n = 4); bars, SD.</p

Exosomes isolated from plasma of high grade glioma patients inhibit CD3+T cells proliferation through an effect mediated by CD14+monocyte.

<p>(A) PBMCs were stimulated with anti-CD3 and anti-CD28 in the absence (white column, CTRL) or presence (black column, GBM-EXO) of different dilutions of exosomes isolated from plasma of high grade glioma patients (GBM). Healthy donor plasma-derived exosomes were used as control (striped column, EXO-HEALTHY). (B) PBMCs or CD14 negatively-sorted PBMCs (CD14-) were stimulated with anti-CD3 and anti-CD28 in the absence (white column, CTRL) or presence (black column, GBM-EXO) of exosomes isolated from plasma of GBM, 1:10 dilution. Proliferation of CD3+ was measured by CFSE assay on day 4. Columns, mean (n = 6); bars, SD;*, significantly different from the control; P<0.05.</p

GSC-derived exosomes are internalized by monocytes and stimulate proliferation of CD14 negatively-sorted PBMCs.

<p>Fluorescent exosomes (A) were incubated with PBMCs for 5 hours and the uptake by CD14+ monocytes, CD4+ or CD8+ T cells was measured by flow cytometric analysis in the bulk population. In representative cytometry histograms, the isotype control is in black and in grey are PBMCs cells incubated with labelled-exosomes and gated on CD14+ monocytes (left, top), on CD4+ (left, bottom) or on CD8+ T cells (right, bottom). (B) Gating and sorting strategies of PBMCs and CD14-depleted PBMC. (Top, left) physical parameters, i.e. forward scatter (FSC) and side scatter (SSC), were used to select PBMCs (gate R1). Monocytes were recognized by evaluating, in PBMCs, the expression of CD14 (gate R2, top, right panel). A PE-isotype matched antibody was used to define R2 (top, central panel). PBMCs-depleted cells were identified as cells included in R1 but not in R2. (C) Representative dot-plots showing the reanalysis of the FACS-sorted PBMCs (left panel) and CD14-depleted PBMCs (right panel). As expected, CD14-positive cells were present in the sorted PBMCs- but not in the CD14-depleted- samples. (D) Proliferation of both unfractioned PBMCs and PBMCs depleted of the CD14+ population (CD14-) was measured, after CFSE labelling assay, by flow cytometry. Cells were pre-incubated without (white column, CTRL) or with (black column, GSC-EXO) GSC-derived exosomes. Columns, mean (n = 6); bars, SD; *, significantly different from the control; P<0.05. Representative cytometry CFSE histograms of PBMCs (C-E) and CD14- cells (D-F) are shown with the percentage of proliferative cells indicated.</p

Influence of methods RWCT on improvement of pupil's comunication skills

The thesis is focused on the programme Reading and Writing for Critical Thinking (RWCT) and its influence on the pupil's communication skills. The theoretical part provides factual information relating to the topic. It describes communication, communication skills, the status of communication in the Framework Education Programme for Primary Education and summarizes the information about the programme RWCT, its objectives, content and methods. The practical part deals with the influence of methods RWCT on improvment of pupil's communicative skills. It compares lessons at two elementary schools in terms of time, which is spended with pupil's communication, at which they have to express their own thoughts and opinions