Journal: bioRxiv

Article Title: Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

doi: 10.1101/2023.01.07.523110

Figure Lengend Snippet: a, Standard scRNA-seq equipment including Sony cell sorter, 10X Chromium scRNA-seq device, and Illumina sequencers are used in the different steps of the SEC-seq workflow (top to bottom). Checkmarks indicate the steps that were validated with corresponding equipment. b, Standard curve of VEGF-A on nanovials using recombinant VEGF-A, immobilized via the VEGF-A capture antibody, and detected with AF647-tagged anti-VEGF-A detection antibody shows a dynamic range across 3 orders of magnitude. Horizontal line represents the detection threshold (see ). c, (left) Schematic showing the steps of the VEGF-A secretion assay in single MSC-loaded nanovials. (middle) Flow cytometry histograms of VEGF-A secretion from single MSCs on nanovials after 0, 6, 12 hours of incubation. (right bottom) VEGF-A secretion assay on single MSC-loaded nanovials with and without VEGF-A capture antibody (Ab). More than 90% of cells in nanovials with capture antibody had fluorescence signal above the threshold (dotted line). (right top) The fluorescence microscopy image shows single MSCs on nanovials with secreted VEGF-A detected with a fluorescently (AF647)-tagged VEGF-A detection antibody (magenta) and cells stained with calcein AM (green). Scale bar is 50 µm. d, Bar plot shows cell viability measured by image analysis of live/dead stain (see ) following flow sorting of cells in suspension or loaded in nanovials. e, (top) Schematic of nanovial loading into droplets with 10X training gel beads in the absence of detergent (to prevent cell lysis). (bottom) Brightfield and fluorescence images of nanovials (red) with single MSCs (green) together with a gel bead in a droplet following emulsification. Scale bar is 50 µm. f , Graph showing the proportion of all nanovial-containing droplets with the indicated number of nanovials. g, After droplet formation and in the presence of lysis buffer, lysis of calcein (green)-labeled cells on nanovials was observed by diffusion of the green fluorescent signal throughout the droplets containing single-MSC loaded nanovials. Overlaid fluorescence and brightfield images of droplets generated with a 10X Genomics NextGEM kit. Scale bar is 50 µm. h, Distribution of species-specific reads from a scRNA-seq experiment with nanovials containing human MSCs or mouse fibroblasts pooled in a 1:1 ratio. Species identity was called by mapping to a joined genome contig and determining the ratio of reads from each species’ genome. i, Comparison of transcripts per cell for either suspended (unsorted) MSCs, suspended and sorted (sorted) MSCs, or MSCs loaded on nanovials and sorted (nanovial). j, Scheme explaining the experiment where MSCs cultured under normoxic and hypoxic conditions, respectively, were loaded on nanovials labeled with different oligo-barcoded streptavidin molecules (‘normoxic’ and ‘hypoxic’ barcodes) and analyzed in a 1:1 ratio in a single 10X channel. The scatter plot below depicts the assignment of cells based on the normoxic or hypoxic oligo-barcode attached to nanovials via streptavidin. Mixed cells have a signal for both barcodes. k , UMAP plots of the scRNA-seq data derived from the experiments in (j), where each cell is labeled by their oligo-barcode assignment. Mixed cells are excluded in UMAPs. l, The UMAP from (k) labeled by the hypoxic gene expression signature to identify MSCs cultured in hypoxic conditions.

Article Snippet: To measure the binding of the oligo-barcoded anti-VEGF-A detection antibodies, we then incubated the four samples with an equal volume of 71.5 ng/ml of AF647 goat anti-mouse IgG (Jackson ImmunoResearch), which binds to the oligo-barcoded Anti-VEGF-A antibody which is mouse species, for 30 minutes at room temperature on a tube rotator.

Techniques: Recombinant, Flow Cytometry, Incubation, Fluorescence, Microscopy, Staining, Suspension, Lysis, Emulsification, Labeling, Diffusion-based Assay, Generated, Comparison, Cell Culture, Derivative Assay, Gene Expression

Journal: bioRxiv

Article Title: Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

doi: 10.1101/2023.01.07.523110

Figure Lengend Snippet: List of reagents and resources

Article Snippet: To measure the binding of the oligo-barcoded anti-VEGF-A detection antibodies, we then incubated the four samples with an equal volume of 71.5 ng/ml of AF647 goat anti-mouse IgG (Jackson ImmunoResearch), which binds to the oligo-barcoded Anti-VEGF-A antibody which is mouse species, for 30 minutes at room temperature on a tube rotator.

Techniques: Conjugation Assay, Recombinant, Modification, Cell Culture, Enzyme-linked Immunosorbent Assay, Sequencing, Software

Journal: Cell

Article Title: An Immune Atlas of Clear Cell Renal Cell Carcinoma

doi: 10.1016/j.cell.2017.04.016

Figure Lengend Snippet:

Article Snippet: Anti-mouse IgG1, 2a, 3 (Fc fragment) - HRP , Jackson Immuno , Cat# 115-035-164.

Techniques: Purification, Recombinant, Electron Microscopy, Isolation, Labeling, Blocking Assay, Mass Cytometry, Software, Diffusion-based Assay

Journal: Nature Communications

Article Title: Galectin-9 binds IgM-BCR to regulate B cell signaling

doi: 10.1038/s41467-018-05771-8

Figure Lengend Snippet: Galectin-9 regulates BCR microcluster formation and signaling. a Representative images of primary naive WT (top) and Gal9-KO B cells (bottom) fixed on bilayers containing anti-kappa as surrogate antigen (Ag) after 90 s of spreading and imaged by confocal microscopy. Brightfield (left) and confocal (right) visualizing antigen mapped to an 8-bit fire color scale (ImageJ). Scale bar 2 μm. Quantification of b area of spreading, c total antigen fluorescence intensity at the cell-bilayer contact, and d mean intensity of antigen for WT (black circles) and Gal9-KO (blue diamonds) cells (each dot represents 1 cell, 200 cells measured per condition), with the mean ± SEM indicated by the red bar, **** p < 0.0001, Mann-Whitney test. Data representative of at least three independent experiments. e – h Primary naive B cells from WT and Gal9-KO mice were settled onto anti-IgM-coated plates for the indicated time. Cells were lysed and subjected to SDS-PAGE followed by immunoblotting with e anti-phosphotyrosine and anti-ERK1/2, f anti-phospho-CD19, g anti-phospho-Akt, and h anti-phospho ERK1/2 (pERK) and anti-β tubulin. Data representative of at least three independent experiments. f Quantification of the fold increase in pCD19, pAkt, and pERK, with the mean ± SEM indicated by bar. Data were analyzed by two-way ANOVA, followed by Sidak’s multiple comparisons test; * p < 0.05, ** p < 0.01

Article Snippet: Primary murine B cells from WT and Gal9-KO mice were labeled with 4 ng/mL Attotec ® 633-labeled goat anti-mouse IgM Fab fragment (Jackson ImmunoResearch, Cat. No. 115-007-020) in 0.5% FBS in PBS for 15 min at 4 °C.

Techniques: Confocal Microscopy, Fluorescence, MANN-WHITNEY, SDS Page, Western Blot

Journal: Nature Communications

Article Title: Galectin-9 binds IgM-BCR to regulate B cell signaling

doi: 10.1038/s41467-018-05771-8

Figure Lengend Snippet: Treatment with exogenous galectin-9 suppresses BCR signaling. a Representative flow cytometric histograms of WT (left) and Gal9-KO (middle) B cells either untreated or treated with various concentrations of recombinant galectin-9 (rGal9; 0.1, 0.2, 0.5, and 1 μM) followed by surface staining for galectin-9 and analyzed using flow cytometry. Overlay of endogenous galectin-9 surface expression in WT cells, and Gal9-KO cells treated with 0.1 μM rGal9 (right). b Naive B cells from WT and Gal9-KO mice treated with 0.1 μM rGal9 were settled onto anti-IgM-coated plates for the indicated time. Cells were lysed and subjected to SDS-PAGE followed by immunoblotting with anti-phospho ERK1/2 and anti-β tubulin (left panel). Quantification of the fold change in pERK over time, averaged over two independent experiments with the mean ± SEM indicated by the bar (right panel). c – f Naive B cells from WT mice were treated with 1 μM rGal9 and settled onto anti-IgM-coated plates for the indicated time. Cells were lysed and subjected to SDS-PAGE followed by immunoblotting with c anti-phosphotyrosine and ERK1/2, d anti-phospho-CD19, e anti-phospho-Akt, and f anti-phospho ERK1/2 and anti-β tubulin. Quantification of the fold change in pCD19, pAkt, and pERK over time, averaged over three independent experiments, with the mean ± SEM indicated by the bar is shown in the right panel. Statistical significance measure by two-way ANOVA followed by Sidak’s multiple comparisons test; **** p < 0.0001, ** p < 0.01, * p < 0.05

Article Snippet: Primary murine B cells from WT and Gal9-KO mice were labeled with 4 ng/mL Attotec ® 633-labeled goat anti-mouse IgM Fab fragment (Jackson ImmunoResearch, Cat. No. 115-007-020) in 0.5% FBS in PBS for 15 min at 4 °C.

Techniques: Recombinant, Staining, Flow Cytometry, Expressing, SDS Page, Western Blot

Journal: Nature Communications

Article Title: Galectin-9 binds IgM-BCR to regulate B cell signaling

doi: 10.1038/s41467-018-05771-8

Figure Lengend Snippet: Galectin-9 alters IgM-BCR nanoclusters. a TIRFM image of surface IgM and fluorescently labeled rGal9 before bleaching for image acquisition (two left panels respectively). dSTORM images reconstructed from single-molecule localization processed by Thunderstorm software mapped to a fire color scale as indicated; the magnified region (3 × 3 µm) from ROI (white box) is shown as 2D image (middle) and 3D surface plot (right) in the order of WT (top), Gal9-KO (middle), and Gal9-KO + 1 µM rGal9 (bottom). Scale bar represents 2 µm. b Quantification of the distribution of IgM by H function and c Hopkins index of localizations inside ROIs. d – g Reconstructed images were analyzed by a model-based Bayesian approach to identify nanoclusters and their physical properties. d Number of clusters (one point per ROI). e Cluster radii (one point per cluster). f Number of molecules (one point per cluster). g Percentage of localization in clusters (one point per ROI). Each category contains at least 15 ROIs from three independent experiments (at least four cells per experiment). Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparison test ( d , e , f ) and one-way ANOVA with Tukey’s multiple comparison test ( b , g ). Red bars indicate mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Primary murine B cells from WT and Gal9-KO mice were labeled with 4 ng/mL Attotec ® 633-labeled goat anti-mouse IgM Fab fragment (Jackson ImmunoResearch, Cat. No. 115-007-020) in 0.5% FBS in PBS for 15 min at 4 °C.

Techniques: Labeling, Software

Journal: Nature Communications

Article Title: Galectin-9 binds IgM-BCR to regulate B cell signaling

doi: 10.1038/s41467-018-05771-8

Figure Lengend Snippet: Galectin-9 immobilizes IgM-BCR and attenuates BCR microclustering. a Diffusion coefficients and b frequency distribution histogram of single-particle tracking of IgM in WT (black circle) or Gal9-KO (blue diamond) primary B cells with the median indicated in red. Five hundred representative diffusion coefficients from a total of at least 1500 tracks from three independent experiments. c Representative TIRF image of fluorescently labeled rGal9 on primary B cell (left) and mask (right) created to differentiate tracks inside Gal9 regions (lower left, red lines) and tracks outside Gal9 regions (lower right, yellow lines). d Diffusion coefficients and e frequency distribution inside Gal9 regions (black circle) and outside Gal9 regions (black triangle) with the median indicated in red. In all, 250 representative diffusion coefficients from a total of at least 900 tracks from three independent experiments. f Representative TIRF microscopy images of WT cell (top) and WT cells treated with 1 μM rGal9 (bottom) on artificial planar lipid bilayers containing anti-kappa mapped to an 8-bit fire color scale (ImageJ). g Quantification of the total antigen intensity at the cell-bilayer interface in WT (black circles) and WT treated with rGal9 (open circles), with the mean ± SEM indicated by the red bar. Scale bar represents 2 μm. Statistical significance assessed by Mann-Whitney; **** p < 0.0001, *** p < 0.001, ** p < 0.01

Article Snippet: Primary murine B cells from WT and Gal9-KO mice were labeled with 4 ng/mL Attotec ® 633-labeled goat anti-mouse IgM Fab fragment (Jackson ImmunoResearch, Cat. No. 115-007-020) in 0.5% FBS in PBS for 15 min at 4 °C.

Techniques: Diffusion-based Assay, Single-particle Tracking, Labeling, Microscopy, MANN-WHITNEY

Journal: Nature Communications

Article Title: Galectin-9 binds IgM-BCR to regulate B cell signaling

doi: 10.1038/s41467-018-05771-8

Figure Lengend Snippet: The galectin-9 lattice increases the molecular density of IgM-BCR and co-receptors. a Representative confocal images of primary WT B cells treated with 1 µM rGal9 and immuostained for CD45 (cyan), IgM (magenta), and galectin-9 (Gal9; yellow). b Fluorescence intensity profile of CD45, IgM, and Gal9 along the cell membrane. c Representative example of masking output of algorithm to detect regions of high galectin-9 (Gal9 high ) and low galectin-9 (Gal9 low ). d Mean fluorescence intensity of CD45 (left) and IgM (right) in Gal9 high and Gal9 low regions. e Representative confocal images of WT B cells treated with 1 µM rGal9 and immunostained for CD22 (cyan), IgM (magenta), and Gal9 (yellow). f Fluorescence intensity profile of CD22, IgM, and Gal9 along the cell membrane. g Mean fluorescence intensity of CD22 (left) and IgM (right) in Gal9 high and Gal9 low regions. h Representative confocal images of WT B cells treated with 1 μM rGal9 and immunostained for CD19, IgM, and Gal9. i Fluorescence intensity profile of CD19, IgM, and Gal9 along the cell membrane. j Mean fluorescence intensity of CD19 (left) and IgM (right) in Gal9 low and Gal9 high regions. Data representative of at least three independent experiments. Each dot represents 1 cell, at least 30 cells measured per condition per experiment. Mean ± SEM indicated by the red bar. Statistical significance assessed by Mann-Whitney; **** p < 0.0001 *** p < 0.001, * p < 0.05. Scale bar 2 μm

Article Snippet: Primary murine B cells from WT and Gal9-KO mice were labeled with 4 ng/mL Attotec ® 633-labeled goat anti-mouse IgM Fab fragment (Jackson ImmunoResearch, Cat. No. 115-007-020) in 0.5% FBS in PBS for 15 min at 4 °C.

Techniques: Fluorescence, MANN-WHITNEY

Journal: Nature Communications

Article Title: Galectin-9 binds IgM-BCR to regulate B cell signaling

doi: 10.1038/s41467-018-05771-8

Figure Lengend Snippet: Galectin-9 increases colocalization between CD22 and IgM in primary B cells. a Representative merged TIRF (top), dSTORM (middle), and dSTORM zoom (bottom) images showing surface CD45 (magenta) and IgM-BCR (green) on primary wild-type (WT) (left) and galectin-9 knockout (Gal9-KO) (right) B cells. dSTORM ROI (3 × 3 μm) is outlined in yellow (middle) and magnified in dSTORM zoom (bottom). b – e Quantification of at least 20 ROIs from WT and Gal9-KO B cells pooled from three independent experiments. b Hopkin’s index showing randomness of CD45 organization (one point per ROI). c H function derived from Riplely’s K showing degree of CD45 clustering. d Mean diameter of CD45 clusters (one point per ROI). e Mean area of CD45 clusters (one point per ROI). f , g Quantification of at least 15 ROIs from WT and Gal9-KO B cells pooled from three independent experiments. f Coordinate-based colocalization (CBC) histograms of the single-molecule distributions of colocalizations between CD45 and IgM. g Nearest-neighbor distance (NND) analysis of the data shown in f . Symbol represents the median NND of all paired single-molecule localizations from one ROI. h Representative merged TIRF and dSTORM images showing surface CD22 (magenta) and IgM-BCR (green) on primary WT (left) and Gal9-KO (right) B cells. i – l Quantification of at least 30 ROIs from WT and Gal9-KO B cells pooled from three independent experiments. i Mean Hopkin’s index showing randomness of CD22 organization (one point per ROI). j H function showing degree of CD22 clustering. k Mean diameter of CD22 clusters (one point per ROI). l Mean area of CD22 clusters (one point per ROI). m , n Quantification of at least 20 ROIs from WT and Gal9-KO B cells pooled from three independent experiments. m CBC histograms of the single-molecule distributions of colocalizations between CD22 and IgM. n NND analysis of the data shown in m . Colocalization between channels shown in white. Scale bars represent 2 and 1 μm (zoom). Mean ± SEM indicated by the red bar. Statistical significance assessed by Mann-Whitney, * p < 0.05

Article Snippet: Primary murine B cells from WT and Gal9-KO mice were labeled with 4 ng/mL Attotec ® 633-labeled goat anti-mouse IgM Fab fragment (Jackson ImmunoResearch, Cat. No. 115-007-020) in 0.5% FBS in PBS for 15 min at 4 °C.

Techniques: Knock-Out, Derivative Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: Galectin-9 binds IgM-BCR to regulate B cell signaling

doi: 10.1038/s41467-018-05771-8

Figure Lengend Snippet: Schematic model of galectin-9 regulation of B-cell activation. a In resting primary naive WT B cells, galectin-9 facilitates interactions between BCRs and either the inhibitory proteins CD45 or CD22 through binding to N-linked glycans, providing a basal attenuation of B-cell signaling upon antigen stimulation. b BCR signaling is enhanced in Gal9-KO B cells due to loss of association of BCR with inhibitory co-receptors. c Treatment of WT B cells with rGal9 induces the association of IgM-BCR with CD45 and CD22 to suppress B-cell signaling

Article Snippet: Primary murine B cells from WT and Gal9-KO mice were labeled with 4 ng/mL Attotec ® 633-labeled goat anti-mouse IgM Fab fragment (Jackson ImmunoResearch, Cat. No. 115-007-020) in 0.5% FBS in PBS for 15 min at 4 °C.

Techniques: Activation Assay, Binding Assay

Journal: International Journal of Biomaterials

Article Title: One-Year Multicenter Randomised Controlled Trial Comparing OT Equator® and Locator Attachments to Retain an Early Loaded Implant Overdenture on Two Implants

doi: 10.1155/2023/2745262

Figure Lengend Snippet: Mean implant length and diameter per group and brand.

Article Snippet: 11.5 , 3 , 11.5 , 3 , Megagen AnyRidge.

Techniques: Control

Journal: International Journal of Biomaterials

Article Title: One-Year Multicenter Randomised Controlled Trial Comparing OT Equator® and Locator Attachments to Retain an Early Loaded Implant Overdenture on Two Implants

doi: 10.1155/2023/2745262

Figure Lengend Snippet: Mean implant length and diameter per group and brand.

Article Snippet: 11.5 , 4 , 11.5 , 4 , Megagen AnyRidge.

Techniques: Control

Journal: Acta Neurochirurgica

Article Title: Methodological and ethical challenges in the use of focused ultrasound for blood–brain barrier disruption in neuro-oncology

doi: 10.1007/s00701-023-05782-5

Figure Lengend Snippet: Summary of unpublished, registered clinical trials on therapeutic focussed ultrasound for BBB modulation

Article Snippet: Carpentier A et al. (2016)[ ] , 1/2a , France , Recurrent GBM , 15 , Humans , Drug : Carboplatin Device : 11.5-mm SonoCloud (CarThera) US device + MRI , 2–6 months.

Techniques: Clinical Proteomics, Sonication, Magnetic Resonance Imaging, Disruption

Journal: Acta Neurochirurgica

Article Title: Methodological and ethical challenges in the use of focused ultrasound for blood–brain barrier disruption in neuro-oncology

doi: 10.1007/s00701-023-05782-5

Figure Lengend Snippet: Summary of main findings of all published original studies on therapeutic FUS for BBB modulation

Article Snippet: Carpentier A et al. (2016)[ ] , 1/2a , France , Recurrent GBM , 15 , Humans , Drug : Carboplatin Device : 11.5-mm SonoCloud (CarThera) US device + MRI , 2–6 months.

Techniques: Activity Assay, Disruption, In Vivo, Sonication, Concentration Assay, Comparison, Control, In Vitro, Biomarker Discovery, Molecular Weight, Animal Model, Inhibition, Injection, Plasmid Preparation, Expressing, Fluorescence, Virus, Produced

Journal: Acta Neurochirurgica

Article Title: Methodological and ethical challenges in the use of focused ultrasound for blood–brain barrier disruption in neuro-oncology

doi: 10.1007/s00701-023-05782-5

Figure Lengend Snippet: Summary of study characteristics and methodology of published original studies on therapeutic FUS for BBB modulation

Article Snippet: Carpentier A et al. (2016)[ ] , 1/2a , France , Recurrent GBM , 15 , Humans , Drug : Carboplatin Device : 11.5-mm SonoCloud (CarThera) US device + MRI , 2–6 months.

Techniques: In Vitro, Ex Vivo, Injection, Imaging, Mouse Assay, Plasmid Preparation, Modification, Recombinant, Diagnostic Assay, Virus, Sonication, Amplification, Magnetic Resonance Imaging