Journal: The Journal of Cell Biology

Article Title: Productive HIV-1 infection of tissue macrophages by fusion with infected CD4 + T cells

doi: 10.1083/jcb.202205103

Figure Lengend Snippet: Related to and . (A–C) Related to . (A) Analysis of Myosin expression and MLC phosphorylation by Western blot: MDMs were transfected with non-targeting siRNA (siCTRL) or targeting Myosin IIA (siMYO). Left: Representative images of Western blot analysis of the expression of Myosin IIA (top) and p-MLC (middle), with actin as loading control (bottom). Right: Quantification of Myosin IIA/actin, normalized to the siRNA control condition ( n = 6 donors, median ± interquartile range). (B) Quantification of p-MLC/actin, normalized to the siCTRL condition ( n = 6 donors, median ± interquartile range). (C) Representative images of actomyosin cytoskeleton of MDMs treated with siCTL or siMYO. F-actin (phalloidin, gray), Myosin IIA (red), and nuclei (DAPI, cyan). Scale bars, 10 µm. (D–G) Related to . (D) Quantification by flow cytometry of infection of MDMs incubated with isotype control (Control) or antibody targeting CD9 (anti-CD9) and co-cultured with infected Jurkat cells, normalized to the isotype control condition ( n = 10 donors, median ± interquartile range). (E) Analysis of CD81 depletion by flow cytometry: MDMs were transfected with non-targeting siRNA (siCTRL) or targeting CD81 (siCD81). Left: Representative dot plot of CD81 signals. Right: Quantification of CD81 expression, normalized to the siCTRL condition ( n = 6 donors, mean ± SD). (F) Flow cytometry analysis of alveolar macrophages infection after a 24 h-co-culture with uninfected Jurkat cells (NI) or Jurkat cells infected for 2 d in presence of anti-CD81 antibody or corresponding isotype control. Representative dot plots of HIV-p24 signals and gating strategy for selection of infected cells. See quantification in . (G) Representative images of actomyosin cytoskeleton of MDMs treated with siCTL or siCD81. F-actin (phalloidin, gray), Myosin IIA (red), and nuclei (DAPI, cyan). (G) This image comes from a mosaic (stitched together by the microscope). Scale bars, 10 µm. Statistical analyses: (A, B, and D) Wilcoxon test and (E) Paired t test. * P ≤ 0.05. Source data are available for this figure: .

Article Snippet: The culture medium was then removed and replaced by a dilution of anti-CD18 (clone TS1/18, mouse IgG1, 302102; Biolegend), anti-CD81 (clone 5A6, mouse IgG1, 349502; Biolegend), anti-CD9 (clone M-L13, mouse IgG1, 555370; BD Biosciences), anti-CD4 (clone Leu3a, mouse IgG1, 344602; Biolegend), or mouse IgG1k isotype control (clone MOPC-21, mouse IgG1, 400102; Biolegend).

Techniques: Expressing, Western Blot, Transfection, Flow Cytometry, Infection, Incubation, Cell Culture, Co-Culture Assay, Selection, Microscopy

Journal: Cell Reports

Article Title: Limited extent and consequences of pancreatic SARS-CoV-2 infection

doi: 10.1016/j.celrep.2022.110508

Figure Lengend Snippet:

Article Snippet: Anti-human CD9 (mouse IgG1, clone SN4 C3-3A2) - 171Yb , Fluidigm , Cat# 3171009B; RRID: AB_2877094.

Techniques: Conjugation Assay, Purification, Blocking Assay, Recombinant, Control, Virus, Saline, Modification, Staining, Library Quantification, Antibody Labeling, Flow Cytometry, Software, Cytometry, Sequencing

Journal: Journal of Extracellular Biology

Article Title: Quantum Dot‐Based Immunolabelling of Extracellular Vesicles and Detection Using Fluorescence‐Based Nanoparticle Tracking Analysis

doi: 10.1002/jex2.70072

Figure Lengend Snippet: Overall workflow of EV immunolabelling and Fl‐NTA analysis. Cells are cultured, and EVs are enriched from the cell culture medium. Then polyethylene glycol (PEG) is added to precipitate EVs, followed by centrifugation to isolate EVs. Quantum dots (QDs) are conjugated to anti‐CD9 and anti‐CD63 antibodies for specific labelling of EV surface markers. EVs are immunolabelled with QD conjugated antibodies, excess dyes are removed through additional washing steps. Finally, labelled EVs are resuspended for further analysis. Labelled EVs are analysed using Sc‐/Fl‐NTA. The analysis includes optimisation of labelling conditions, size analysis and comparative studies on different fluorophores, EV markers and cell lines.

Article Snippet: To compare between QD and organic dyes, Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD63 antibody (IgG1, clone#: H5C6, Cat#: 353037, BioLegend) and Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD9 antibody (IgG2B, clone#: 209306, Cat#: FAB1880G, R&D Systems) were also used.

Techniques: Cell Culture, Centrifugation

Journal: Journal of Extracellular Biology

Article Title: Quantum Dot‐Based Immunolabelling of Extracellular Vesicles and Detection Using Fluorescence‐Based Nanoparticle Tracking Analysis

doi: 10.1002/jex2.70072

Figure Lengend Snippet: Characterisation of EVs derived from A549 cells. (A) Sc‐NTA indicating the size distribution of A549‐derived EVs, with the majority falling within the 30–200 nm range. (B) Western blot demonstrating the enrichment of EV‐specific markers, including CD9, CD63 and Hsp70, in EVs isolated from A549 cells, thereby confirming the successful isolation of EVs. (C) SEM image showing the morphology and size of EVs derived from A549 cells, revealing their characteristic spherical shape and nanoscale size.

Article Snippet: To compare between QD and organic dyes, Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD63 antibody (IgG1, clone#: H5C6, Cat#: 353037, BioLegend) and Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD9 antibody (IgG2B, clone#: 209306, Cat#: FAB1880G, R&D Systems) were also used.

Techniques: Derivative Assay, Western Blot, Isolation

Journal: Journal of Extracellular Biology

Article Title: Quantum Dot‐Based Immunolabelling of Extracellular Vesicles and Detection Using Fluorescence‐Based Nanoparticle Tracking Analysis

doi: 10.1002/jex2.70072

Figure Lengend Snippet: Optimisation of EV immunolabelling conditions. Evaluated by varying (A) antibody dilution, (B) incubation time and (C) staining volumes. According to Nanodrop measurements, the concentrations of the antibodies were as follows: for anti‐CD9 antibody corresponds to 0.90 ± 0.02 µM, and for anti‐CD63 antibody corresponds to 1.90 ± 0.20 µM. Immunolabelling efficiency is represented as the ratio of the number concentration in fluorescence mode to that in light scatter mode, serving as an indicator of labelling success. The lines represent B‐spline interpolation, applied to the experimental data points to visualise the overall trend in labeling efficiency. Error bars represent SD ( n = 3).

Article Snippet: To compare between QD and organic dyes, Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD63 antibody (IgG1, clone#: H5C6, Cat#: 353037, BioLegend) and Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD9 antibody (IgG2B, clone#: 209306, Cat#: FAB1880G, R&D Systems) were also used.

Techniques: Incubation, Staining, Concentration Assay, Fluorescence, Labeling

Journal: Journal of Extracellular Biology

Article Title: Quantum Dot‐Based Immunolabelling of Extracellular Vesicles and Detection Using Fluorescence‐Based Nanoparticle Tracking Analysis

doi: 10.1002/jex2.70072

Figure Lengend Snippet: Comparative analysis of size distribution in scatter and fluorescence mode. The NTA plots show the comparison of size distribution of EVs under different conditions: unlabelled EVs measured in light scatter mode (black line), labelled EVs measured in fluorescence mode (blue line) and labelled EVs measured in light scatter mode (red line). Panels (A–C) represent data for CD9‐QD625, while panels (D–F) represent data for CD63‐QD625. All error bars represent SE ( n = 3). (G, H) Stacked bar plots illustrate the percentage distribution of EV sizes in 50 nm intervals for both fluorescence and scatter modes, emphasising differences in EV subpopulations across the size ranges.

Article Snippet: To compare between QD and organic dyes, Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD63 antibody (IgG1, clone#: H5C6, Cat#: 353037, BioLegend) and Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD9 antibody (IgG2B, clone#: 209306, Cat#: FAB1880G, R&D Systems) were also used.

Techniques: Fluorescence, Comparison

Journal: Journal of Extracellular Biology

Article Title: Quantum Dot‐Based Immunolabelling of Extracellular Vesicles and Detection Using Fluorescence‐Based Nanoparticle Tracking Analysis

doi: 10.1002/jex2.70072

Figure Lengend Snippet: Identification of EVs subpopulations from various cell lines and their relative abundance. (A) Comparative size distribution profiles of CD9‐(+) (black line) and CD63‐(+) (red line) EV populations derived from EA.hy926, THP‐1 and A549 cells, measured in fluorescence mode. Error bars represent SE ( n = 3). (B) Bar graph shows relative abundance of CD9‐positive and CD63‐positive EVs as a percentage, with error bars indicating SD ( n = 3). Note that relative abundance was calculated as the number concentration ratio of FM to LSM.

Article Snippet: To compare between QD and organic dyes, Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD63 antibody (IgG1, clone#: H5C6, Cat#: 353037, BioLegend) and Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD9 antibody (IgG2B, clone#: 209306, Cat#: FAB1880G, R&D Systems) were also used.

Techniques: Derivative Assay, Fluorescence, Concentration Assay

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: a Western blot showing transmembrane proteins (CD9, CD63, and CD81), a cytosolic protein (syntenin-1) and two “negative” controls (AChE and calnexin) in cell lysates (CL) and the pellets obtained from HeLa 24 h conditioned medium after differential ultracentrifugation (2 K, 10 K, 200 K). The loaded material comes from 20 × 10 6 cells for the centrifugation pellets, and from 0.2 × 10 6 cells for the cell lysate. Representative of 3 independent experiments. b NTA and EM analysis of 200 K pellets obtained from HeLa 24 h conditioned medium. The quantifications represent the mean of concentration or frequency of EVs of different diameters, error bars show the standard deviation (SD). N = 3 independent experiments. Scale bar of the zooms: 0.1 μm. c Principle of immunoprecipitation of CD63 and CD9 EVs in HeLa concentrated conditioned medium and representative Western blot of the pull-down (PD) and flow-through (FT) of the immunoprecipitation, with quantification of the relative CD63 and CD9 bands intensity of three independent experiments (mean ± SD is represented). d Immuno-EM analysis of the 200 K pellet labeled with anti-CD9 (5 nm gold particles) and anti-CD63 (10 nm gold particles) antibodies. This experiment was performed once. Scale bar of the zooms: 0.1 μm. e Confocal imaging of immunofluorescence staining of CD63 and CD9 in HeLa cells. Pink arrows show CD9 localized in intracellular compartments. Representative picture of two independent experiments. Scale bar: 5 μm.

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Western Blot, Centrifugation, Concentration Assay, Standard Deviation, Immunoprecipitation, Labeling, Imaging, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: a Principle of the RUSH system used to follow CD63 and CD9 intracellular trafficking. SBP streptavidin binding peptide, strept streptavidin, ER endoplasmic reticulum. b Micrographs and quantifications of live imaging of HeLa cells co-transfected with the CD63-mCherry and CD9-eGFP RUSH plasmids. Biotin at 40 μM was added at T = 0. White arrows show peripheral compartments where CD63 and CD9 co-localize. Z -projection of 11 planes. Scale bar: 5 μm. Quantification upon time of three independent experiments showing the mean ± SD eGFP and mCherry fluorescence intensity in the Golgi and in large compartments, the mean ± SD number of eGFP- or mCherry-positive small compartments and the median and range of the Pearson’s co-localization coefficient between eGFP and mCherry where automatically quantified. N = 3 independent experiments. 5 fields per experiments where imaged, for a total of at least 10 individual cells to analyze per experiment. c Representative electron microscopy images of HeLa cells co-transfected with RUSH constructs of CD63-mCherry and CD9-eGFP , 1 h or 2 h after incubation with biotin, or at steady-state, labeled with anti-eGFP gold 10 nm (red arrows) and anti-mCherry gold 15 nm (blue arrows). Relative labeling index (RLI) in each compartment quantified from 7 different fields per replicate is represented as mean ( n = 2 independent biological replicates). d Confocal microscopy pictures of HeLa cells ( z -projection) co-transfected with CD63-mCherry- and CD9-eGFP- RUSH plasmids, and stained with anti-Rab7 after 1 h of incubation with biotin. Scale bar: 10 μm. Mander’s coefficients representing the % of CD9 + /CD63-, CD9-/CD63 + , and CD9 + /CD63 + intracellular compartments also positive for the Rab7 signal in each cell are shown. Results from two independent experiments are shown, each dot represents one cell (23 cells from replicate 1, and 24 cells from replicate 2) and the median is represented. Ordinary one-way ANOVA with a Tukey’s multiple comparisons test was performed to compare the different categories of intracellular compartments shown on the graph.

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Binding Assay, Imaging, Transfection, Fluorescence, Electron Microscopy, Construct, Incubation, Labeling, Confocal Microscopy, Staining

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: a Scheme of the structure and C-terminal sequences of CD63-WT and the mutant CD63-YA. b Immunofluorescence of HeLa cells transfected with the RUSH CD63-eGFP or CD63-YA-eGFP plasmids at steady state. Scale bar 10 μm. This experiment was performed once. c Micrographs of HeLa cells co-transfected with CD63-YA-eGFP and CD63-WT-mCherry or CD9-eGFP and CD63-YA-mCherry , and median ± range of the Pearson’s co-localization coefficient over time between eGFP and mCherry. Biotin was added at T = 0. Z -projection of 11 planes. Scale bar 5 μm. CD63/CD63-YA: 3 independent experiments n = 51 cells, CD9/CD63-YA: 2 independent experiments n = 33 cells. 5 fields per experiment where imaged, for a total of at least 10 individual cells to analyze per experiment.

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Mutagenesis, Immunofluorescence, Transfection

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: a Principle and flow cytometry analysis of anti-GFP surface staining of HeLa cells transfected with the RUSH constructs CD63-WT-eGFP , CD63-YA-eGFP , or CD9-eGFP after different incubation times with biotin followed by fixation. The ratio of the surface staining (AF647) over the total GFP signal mean fluorescence intensities is represented at different time points, time 0 subtracted, mean ± SD for 3 independent experiments. b Principle and flow cytometry analysis of anti-GFP uptake after surface staining of HeLa cells transfected with the RUSH constructs CD63-WT-eGFP , CD63-YA-eGFP , or CD9-eGFP after 2 h of incubation with biotin. The mean percentage ± SD of internalized anti-GFP-AFP647 is represented for 3 independent experiments. Ordinary one-way ANOVA, Tukey’s multiple comparisons test. Gating strategy is illustrated in Supplementary Fig. .

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Flow Cytometry, Staining, Transfection, Construct, Incubation, Fluorescence

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: a Western blot of the cell lysate (CL) and of the different EV pellets obtained by differential ultracentrifugation of CCM from HeLa cells transfected with the RUSH plasmids CD63-WT-eGFP or CD63-YA-mCherry (24 h release with biotin). EVs from 20 × 10 6 cells and CL from 0.2 × 10 6 cells were loaded. The intensity of the band corresponding to the mCherry fusion proteins was quantified and normalized by the intensity of the CD9 band in 3 independent experiments, the mean ± SD is represented. Two-tailed paired t test. b Representative Western blot of the pull-down (PD) and flow-through (FT) of the immunoprecipitation of EVs from HeLa cells transfected with the RUSH plasmids CD63-WT-eGFP , CD63-YA-eGFP , or CD9-eGFP , recovered 24 h after biotin addition. 60 × 10 8 total particles quantified by NTA were used for each IP. Percent of GFP + cells quantified by flow cytometry were similar in the three conditions (Supplementary Fig. ). The GFP bands intensity in the PD normalized to endogenous CD9 in the corresponding PD are represented as mean ± SD for 3 independent experiments. Ordinary one-way ANOVA, Tukey’s multiple comparisons test. c Representative Western blot of EVs (200 K pellets) from HeLa cells transfected with the CD63-WT , CD63-YA , or CD9-eGFP RUSH plasmids treated with DMSO of BafA1 100 nM during 16 h. The same number of EVs between the DMSO and the BafA1 conditions were loaded on the gel (around 100 × 10 8 particles). The fold change between DMSO and BafA1 treatment for each construct is represented as mean ± SD for 3 independent experiments. Two-tailed one sample t test to compare each condition with a theoretical mean of 1. d Proportion of cellular endogenous CD9 and CD63 released in EVs, as semi-quantified on Western blots. The signal for CD9 and CD63 in 200 K pellets released by 20 × 10 6 HeLa cells was divided by the signal for the same molecule in the total lysate of 0.2 × 10 6 cells, run on the same blot. 1 representative Western blot and quantification (mean ± SD) of 3 independent experiments. Two-tailed paired t test.

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Western Blot, Transfection, Two Tailed Test, Immunoprecipitation, Flow Cytometry, Construct

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: EVs were isolated by anti-GFP immuno-isolation from either non-transfected HeLa cells, or HeLa transfected with CD63-eGFP -RUSH or CD9-eGFP -RUSH, either 3 h or 24 h after biotin addition, and their composition was analyzed by mass-spectrometry. a Volcano plots representing quantified proteins with at least 2 peptides in 2 replicates in at least one condition. Shown are the fold changes of peptide abundancy between CD63- and CD9-eGFP expressing EV samples and the p -value of this quantification, for EVs recovered 3 h (left) or 24 h (right) after biotin addition. Position of the membrane-associated proteins selected for further analysis is indicated. b Results of the FunRich gene enrichment analysis among the proteins either enriched in the CD63- (blue), or CD9-eGFP (red) samples, or common between the CD63 and CD9-eGFP samples (purple) at 3 h or 24 h after biotin addition. For each subcellular compartment protein list, % of proteins of this category in the list of CD63-, CD9-, or common CD63/CD9 proteins is indicated, and the p -value of this percentage being different to its counterpart in the whole HeLa cell database is calculated. P -value (hypergeometric uncorrected). c Schematic representation of the transmembrane proteins identified in the CD63-, CD9-, or CD63/CD9-eGFP EVs at 3 h and 24 h.

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Isolation, Transfection, Mass Spectrometry, Expressing, Membrane

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: Names of proteins identified in EVs and used for the endosome, lysosome, and PM (plasma membrane) categories are listed.

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Clinical Proteomics, Membrane

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: a Western blot showing CD9, CD63, and CD81, and the new markers LAMP1, BSG, and SLC3A2 in cell lysates (CL) and the pellets obtained from HeLa conditioned media after differential ultracentrifugation (2 K, 10 K, and 200 K). The loaded material comes from 20 × 10 6 cells for the centrifugation pellets, and from 0.2 × 10 6 cells for the cell lysate. One representative image. For each marker, mean ± SD of the quantification of the signal in 200 K pellets divided by the signal in the total lysate, run on the same blot, is shown for 3 independent experiments. Ordinary one-way ANOVA, Tukey’s multiple comparisons test. b Viability of HeLa cells at the end of the 16 h medium conditioning period in the presence of DMSO (control) or BafA1 (100 nM) or GW4869 (10 μM) drugs, measured by trypan blue in 6 independent experiments, mean ± SD is represented. No significant difference observed with an ordinary one-way ANOVA, Tukey’s multiple comparisons test. c Nanoparticle tracking analysis (NTA) of EVs obtained by differential ultracentrifugation from equal numbers of HeLa cells treated with DMSO (control), BafA1 or GW4869 during 16 h. The particles concentration according to their size and the fold change of the total particle concentration between treated and control conditions are represented as mean ± SD of 5 (200 K) or 3 (10 K) independent experiments. Two-tailed one sample t test to compare each condition with a theoretical mean of 0. d TEM analysis (1 representative image) and size measurement (in 3 independent experiments) of EVs in 200 K pellets of cells exposed to DMSO, BafA1 or GW4869. Mean ± SD of the frequency distribution of CD63 and CD9 in EVs of different diameters is represented. e Representative Western blot of cell lysates from 0.2 × 10 6 HeLa cells and EVs from 20 × 10 6 HeLa cells treated with DMSO, BafA1, or GW4869, corresponding to the samples of b – d . The mean fold change ± SD between DMSO and BafA1 or GW4869 treatment of the bands intensity in the 200 K and 10 K pellets divided by the cell lysate is represented for 6 independent experiments. Two-tailed one sample t test to compare each condition with a theoretical mean of 0.

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Western Blot, Centrifugation, Marker, Control, Concentration Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: Micrographs of live imaging of HeLa cells co-transfected with either CD63-mCherry or CD9-mCherry and CD81-eGFP RUSH plasmids. Biotin was added at T = 0. The median ± range of the Pearson’s co-localization coefficient between eGFP and mCherry is represented over time after biotin addition. Scale bar: 10 μm. 2 independent experiments. 5 fields per experiment where imaged, for a total of at least 10 cells to analyze per experiment.

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Imaging, Transfection

Journal: bioRxiv

Article Title: Close homologue of L1 sensitizes lung cancer cells to cisplatin and paclitaxel via inhibition Akt pathway

doi: 10.1101/747238

Figure Lengend Snippet: (A) Cell survival of A549 and A549 resistant cells (A549/DDP, A549/PTX) treated with increasing concentration of DDP and PTX were measured by MTT assay. (B) The IC 50 values of DDP in A549/DDP, A549 cells, and the IC 50 values of PTX in A549/PTX, A549 cells. (C) Western blot assays dectected the expression of drug resistance-related genes MDR1, MRP and LRP in A549 cells and A549 resistant cells (A549/DDP, A549/PTX). The expression of CHL1 expression in A549 cells and A549 resistant cells (A549/DDP, A549/PTX) were analysed by Western blot (D) and qRT-PCR assays (E) respectively. (F) The expression of CHL1 mRNA in GSE21656 data. All results were expressed as mean ± SD, and were performed in triplicate. * P < 0.05.

Article Snippet: After blocked by 5% BSA for 2 h at 4°C, the membrane was incubated with primary antibodies anainst CHL1 (1:500, proteintech), MDR1 (1:500, proteintech), MRP (1:500, proteintech), LRP(1:500, proteintech), phospho-Akt(1:1000, Abcam), Akt (1:2000, Abcam), overnight at 4°C.

Techniques: Concentration Assay, MTT Assay, Western Blot, Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Opportunities and Pitfalls of Fluorescent Labeling Methodologies for Extracellular Vesicle Profiling on High-Resolution Single-Particle Platforms

doi: 10.3390/ijms221910510

Figure Lengend Snippet: Characterization of purified sEV subpopulations based on their surface markers. ( A ) Antibody staining efficiency was evaluated by nFCM using anti-CD9 antibodies conjugated with different fluorophores. ( B ) HT29 and HEK293 sEVs were stained with anti-tetraspanin (CD9, CD63, and CD81) antibodies, conjugated with either PE or AF488, and analyzed on nFCM ( n ≥ 3; mean ± SEM). ( C ) Cross-platform and inter-batch variability was assessed by single-staining HT29 sEV (batches #A and #B) with anti-tetraspanin AF488 antibodies ( n = 3; mean ± SEM). Differences in tetraspanin expression between nFCM and F-NTA, and batches #A and #B, were assessed using two-way ANOVA with Tukey’s test for multiple comparisons (alpha = 0.05, p = 0.1234 (ns), 0.0002 (***), <0.0001 (****)). Detailed results of the statistical analysis are provided as . ( D ) To evaluate single, as well as co-expressing, events, HT29 sEVs were stained with a mix of 2 and 3 different anti-tetraspanin AF488 antibodies and analyzed on F-NTA ( n = 3; mean ± SEM), or on ( E ) nFCM using PE-conjugated antibodies ( n = 3; mean ± SEM). HT29 sEV subpopulations expressing either 1, 2, or 3 markers are represented in the Venn diagram on the left. The Venn diagram on the right refers to the same HT29 sEVs, however it depicts subpopulations co-expressing both 2 or 3 markers simultaneously. Additional data from procedural controls, as well as the F-NTA PSD histograms, are provided in .

Article Snippet: The following fluorescently-labeled primary antibodies were used: Phycoerythrin (PE)-conjugated mouse anti-human CD9, CD63 and CD81 (dilution 1:10 for all; Exbio, Vestec, Czech Republic), Alexa Fluor ® 488 (AF488)-conjugated mouse anti-human CD9, CD63 and CD81 (1:500, 1:25 and 1:500, respectively; R&D Systems, Minneapolis, MN, USA), allophycocyanin (APC)-conjugated mouse anti-human CD9 (1:10; Exbio), Alexa Fluor ® 647 (AF647)-conjugated mouse anti-human CD9 (1:10; Exbio).

Techniques: Purification, Staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Opportunities and Pitfalls of Fluorescent Labeling Methodologies for Extracellular Vesicle Profiling on High-Resolution Single-Particle Platforms

doi: 10.3390/ijms221910510

Figure Lengend Snippet: Suitability of SEC and UF as methods for clearing dyes in excess after sEV fluorescent labeling and their effect on subpopulation ratios. ( A ) Comparison of labeling % for stained HT29 sEVs before and after the removal of excess dye. 1 × 10 9 or 5.5 × 10 9 sEVs were incubated with antibodies (aCD9 1:12.5; aCD63 1:12.5; aCD81 1:25) or CFSE (50 µM), respectively, and measured by F-NTA. To remove the excess dye, UF washing strategy was applied, followed by F-NTA detection. ( B ) To compare different strategies for the removal of fluorescent antibodies in excess, labeling %, ( C ) PSD histograms, and ( D ) median and mean particle diameter were assessed for CD9+, CD63+ and CD81+ HEK293 sEVs, on nFCM. 2 × 10 9 sEVs, at a concentration of 10 8 particles/µL, were incubated with PE-labelled antibodies (aCD9 1:500; aCD63 1:25; aCD81 1:500) and unbound antibodies were removed by SEC or UF. Sample dilution (500–1000-fold) served as staining reference. Data is presented as mean ± SEM of at least three independent experiments. F-NTA PSD histograms of samples analyzed before and after washing are provided in .

Article Snippet: The following fluorescently-labeled primary antibodies were used: Phycoerythrin (PE)-conjugated mouse anti-human CD9, CD63 and CD81 (dilution 1:10 for all; Exbio, Vestec, Czech Republic), Alexa Fluor ® 488 (AF488)-conjugated mouse anti-human CD9, CD63 and CD81 (1:500, 1:25 and 1:500, respectively; R&D Systems, Minneapolis, MN, USA), allophycocyanin (APC)-conjugated mouse anti-human CD9 (1:10; Exbio), Alexa Fluor ® 647 (AF647)-conjugated mouse anti-human CD9 (1:10; Exbio).

Techniques: Labeling, Comparison, Staining, Incubation, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Opportunities and Pitfalls of Fluorescent Labeling Methodologies for Extracellular Vesicle Profiling on High-Resolution Single-Particle Platforms

doi: 10.3390/ijms221910510

Figure Lengend Snippet: Multiplex fluorescence analysis for enhanced sEV identification and characterization. ( A ) Purified HT29 sEVs were labeled with anti-CD9-AF488 and anti-CD81-PE either individually (single staining) or in combination (double staining). ( B ) HT29 sEVs were stained with CTR and anti-CD81-AF488 either individually or in combination. Results are shown as % of labeled particles detected on nFCM, in two fluorescence channels ( n = 3; mean ± SEM).

Article Snippet: The following fluorescently-labeled primary antibodies were used: Phycoerythrin (PE)-conjugated mouse anti-human CD9, CD63 and CD81 (dilution 1:10 for all; Exbio, Vestec, Czech Republic), Alexa Fluor ® 488 (AF488)-conjugated mouse anti-human CD9, CD63 and CD81 (1:500, 1:25 and 1:500, respectively; R&D Systems, Minneapolis, MN, USA), allophycocyanin (APC)-conjugated mouse anti-human CD9 (1:10; Exbio), Alexa Fluor ® 647 (AF647)-conjugated mouse anti-human CD9 (1:10; Exbio).

Techniques: Multiplex Assay, Fluorescence, Purification, Labeling, Staining, Double Staining

Journal: Advanced materials (Deerfield Beach, Fla.)

Article Title: Ultrafast and Controlled Capturing, Loading, and Release of Extracellular Vesicles by a Portable Microstructured Electrochemical Fluidic Device.

doi: 10.1002/adma.202212000

Figure Lengend Snippet: Figure 3. Characterization of the purified EVs. A,B) Western blot analysis. A) EVs separated from urine using either anti-CD9 (second lane) or anti-CD63 (third lane) immunoaffinity layer contained the protein markers CD9 (membrane protein) and TSG101 (soluble) and were largely devoid of albumin in comparison with raw urine (first lane). B) EVs separated from serum using either anti-CD9 (second lane) or anti-CD63 (third lane) contained CD9 and TSG101. IgG was used as a protein marker that is highly abundant in serum. C) SEM image of a field of urinary EVs purified using a device with an anti- CD63 immunoaffinity layer. Scale bar: 2 μm. D) Low magnification TEM image of a field of EVs separated from urine with an anti-CD63 immunoaffinity layer and negatively stained with uranyl acetate. Scale bar: 500 nm. E) SEM image of a field of serum EVs purified using a device with an anti-CD9 immunoaffinity layer. Scale bar: 500 nm. F) Large magnification cryo-TEM image of a single EV purified from urine with an anti-CD9 immunoaffinity layer. Scale bar: 100 nm. G) Large magnification cryo-TEM image of a single EV purified from human plasma with an anti-CD9 immunoaffinity layer. Scale bar: 100 nm. H–I) Enzymatic activity of EVs proteins after further purification with the device. EVs were isolated from HS-5 cell culture supernatant by ultracentrifugation, then purified again either with anti-CD73, *p = 0.016 (H) or with anti-CD9 modified device (I) (mean ± SD, n = 3). CD73 enzymatic activity was measured by the addition of malachite green.[38]

Article Snippet: Mouse monoclonal IgG1 κ CD9 antibody (C-4) (sc-13118), mouse monoclonal IgG1 κ CD63 antibody (MX-49.129.5) (sc-5275), and mouse monoclonal IgG1 κ CD9 (C-4) Alexa Fluor® 647 (sc-13118) were purchased from Santa Cruz.

Techniques: Western Blot, Membrane, Comparison, Marker, Staining, Clinical Proteomics, Activity Assay, Isolation, Cell Culture

Journal: Advanced materials (Deerfield Beach, Fla.)

Article Title: Ultrafast and Controlled Capturing, Loading, and Release of Extracellular Vesicles by a Portable Microstructured Electrochemical Fluidic Device.

doi: 10.1002/adma.202212000

Figure Lengend Snippet: Figure 4. A,B,D,E,G,H) NTA plots of purified EVs separated with anti-CD9 (dashed cyan line) or anti-CD63 (green line) modified device from urine (A), cell culture (D), and plasma (G), vs separation from the corresponding physiological fluid by ultracentrifugation (B,E,H). C,F,I) The percentage of particles <200 nm isolated from urine (***p = 0.0005 UC vs Anti-CD9, **p = 0.003 Anti-CD9 vs Anti-CD63) (C), cell-culture medium (****p = 0.000001 UC vs Anti-CD9, ****p = 0.000005 UC vs Anti-CD63, **p = 0.001 Anti-CD9 vs Anti-CD63) (F), and plasma (***p = 0.0002 UC vs Anti-CD9, ****p = 0.000002 UC vs Anti-CD63, ****p = 0.00008 Anti-CD9 vs Anti-CD63) (I) using ultracentrifugation or a device modified with anti-CD9 or anti- CD63 antibodies (mean ± SD, n = 3). All data were smoothed by moving average trendline (period = 4).

Article Snippet: Mouse monoclonal IgG1 κ CD9 antibody (C-4) (sc-13118), mouse monoclonal IgG1 κ CD63 antibody (MX-49.129.5) (sc-5275), and mouse monoclonal IgG1 κ CD9 (C-4) Alexa Fluor® 647 (sc-13118) were purchased from Santa Cruz.

Techniques: Cell Culture, Clinical Proteomics, Isolation