Journal: International Journal of Molecular Sciences

Article Title: Magnetofluoro-Immunosensing Platform Based on Binary Nanoparticle-Decorated Graphene for Detection of Cancer Cell-Derived Exosomes

doi: 10.3390/ijms23179619

Figure Lengend Snippet: Tetraspanin characterization of PC-exosomes: ( A ) concentration of LF-exos and FL images of a dye-tetraspanin-labeled exosome ( B ) on a CD81 capture spot and ( C ) on a CD9 capture spot.

Article Snippet: ExoView Tetraspanin Kits containing tetraspanin antibody dyes (AF488-anti CD9 and AF555-anti CD81) were obtained from NanoView Biosciences (Boston, MI, USA).

Techniques: Concentration Assay, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Magnetofluoro-Immunosensing Platform Based on Binary Nanoparticle-Decorated Graphene for Detection of Cancer Cell-Derived Exosomes

doi: 10.3390/ijms23179619

Figure Lengend Snippet: FL images after LF-exo conjugation with Ag/IO-GRP and AF488-CD9 via antibody and exosome binding: ( A ) bright field image, ( B ) FL image, and ( C ) bright field and FL merged condition image.

Article Snippet: ExoView Tetraspanin Kits containing tetraspanin antibody dyes (AF488-anti CD9 and AF555-anti CD81) were obtained from NanoView Biosciences (Boston, MI, USA).

Techniques: Conjugation Assay, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Magnetofluoro-Immunosensing Platform Based on Binary Nanoparticle-Decorated Graphene for Detection of Cancer Cell-Derived Exosomes

doi: 10.3390/ijms23179619

Figure Lengend Snippet: Disease-specific exosome detection performance based on a magnetofluoro-immunoassay using Ag/IO-GRP and AF488-CD9: ( A ) sensitivity test and ( B ) selectivity test against other exosomes.

Article Snippet: ExoView Tetraspanin Kits containing tetraspanin antibody dyes (AF488-anti CD9 and AF555-anti CD81) were obtained from NanoView Biosciences (Boston, MI, USA).

Techniques:

Journal: Blood Advances

Article Title: Polyphosphate expression by cancer cell extracellular vesicles mediates binding of factor XII and contact activation

doi: 10.1182/bloodadvances.2021005116

Figure Lengend Snippet: Characterization of EV. Particle numbers (A) and relative protein concentrations (B) in the fractions eluted following qEV size-exclusion chromatography. Data are mean ± SEM. (C) Tetraspanin (CD9, CD63, and CD81) content of purified EVs was analyzed in the indicated elution fractions by immunoblotting. (D) Size distribution of pooled fractions 7 to 10 from HDFs (left panel) and L3.6 cells (right panel). (E) Electron microscopy image of EV from HDFs (left panel) and L3.6 cells (right panel).

Article Snippet: Anti-human CD81-AF488 antibody (20 μg/mL, FAB4615G; R&D Systems) was used as a control ligand.

Techniques: Size-exclusion Chromatography, Purification, Western Blot, Electron Microscopy

Journal: Blood Advances

Article Title: Polyphosphate expression by cancer cell extracellular vesicles mediates binding of factor XII and contact activation

doi: 10.1182/bloodadvances.2021005116

Figure Lengend Snippet: polyP on EV derived from cancer cells mediates ligand binding. (A) The amount of polyP associated with cancer cell–derived EV was estimated by comparison with a standard curve (supplemental Figure 2A). (B) Detection of polyP on L3.6-derived EVs following incubation with buffer (HBS), DNase I and RNase A (D/R), or CIP. polyP of different chain lengths was used to estimate the size of EV-associated polyP. L3.6-derived EVs were preincubated with buffer or E coli PPX, and the binding of PPX-Δ12–AF488 (C), anti-CD81–AF488 (D), and or FXII-AF488 (E) was measured. The number of total EV was determined in scatter mode, and the number of ligand-binding EV was determined in fluorescence mode using a 488-nm excitation laser. The differences in fluorescent particle numbers were compared by estimating the area under each curve (AUC) using GraphPad Prism 8. Bars represent means ± SEM. *** P < .001, 1-way ANOVA with multiple comparisons. L, long; M, medium; S, short.

Article Snippet: Anti-human CD81-AF488 antibody (20 μg/mL, FAB4615G; R&D Systems) was used as a control ligand.

Techniques: Derivative Assay, Ligand Binding Assay, Comparison, Incubation, Binding Assay, Fluorescence

Journal: bioRxiv

Article Title: Single-cell RNA sequencing identifies phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow

doi: 10.1101/2022.01.26.477664

Figure Lengend Snippet: (A) Dot plot of surface marker gene expression in different stromal cell clusters. Cluster numbers and corresponding stromal cell groups are indicated on the y-axis legend. Dot sizes represent the percentage of cells expressing a certain gene in each cluster and dot colors represent the scaled average expression of that gene. (B) FACS plots illustrating the gating strategy for the isolation of different stromal subsets. The displayed cell populations are indicated on top of the plot. Following exclusion of doublets, dead cells, CD45- and CD235a-expressing cells, CD45 low/- CD235a - CD71 - CD271 + cells (left panel) were gated based on CD52 and NCAM1 expression (middle panel). The resulting three populations were labelled as A-C (middle panel), corresponding to the stromal cell groups in (A) (A, CD52 - NCAM1 - ; B, CD52 - NCAM1 + ; C, CD52 + NCAM1 - ). CD45 - CD235a - CD71 - CD271 + CD52 - NCAM1 - (group A) cells were further divided based on CD81 expression and four populations were identified (A1-A4) (right panel). A1, CD81 ++ ; A2, CD81 + ; A3; CD81 +/- ; A4, CD81 - . (C) CFU-F frequencies of sorted stromal cell populations as shown in (B). Data are presented as individual data (dots) and median (horizontal lines) from independent experiments (n=3-6). Symbol colors and x-axis labels correspond to the cell population colors in (B). *: p<0.05; ****: p <0.0001 (Kruskal-Wallis test). (D) In vitro differentiation capacity of sorted stromal cell populations (as indicated in B) towards the adipogenic, osteoblastic, and chondrogenic lineage. Non-induction controls are shown in the left panel. Scale bars represent 200 µm. A representative set of pictures from a total of three independent experiments is shown. (F) Formalin-fixed, paraffin-embedded (FFPE) human BM slides were sequentially stained for DAPI (blue), CD45 (yellow), CD81 (green), CD271 (pink), and NCAM1 (cyan) and scanned with the OlympusVS120 slide scanner. Different staining combinations are shown as indicated under each picture to provide better visualization of individual staining obtained from the same FFPE slide. Scale bars represent 50 µm. Red arrows: CD271 + CD81 ++ cells; white arrows: arteriolar walls; white lines: bone lining regions. Bone (b), adipocytes (a), and capillaries (*) are indicated.

Article Snippet: The following antibodies were used for FACS analysis and cell sorting: CD235a-PE-Cy5 [clone GA-R2 (HIR2)], CD71-PE-Cy5 (clone M-A712), CD45-APC-Cy7 (clone 2D1), CD52-Alexa Fluor (AF) 488 (clone 4C8), CD56 (NCAM1)-Brilliant Violet 605 (clone Leu-19, NKH1), CD81-PE (JS-81) (all BD Bioscience), LEPR-PE (clone 52263, R&D Systems) CD271-APC (clone ME20.4-1.H4, Miltenyi).

Techniques: Marker, Expressing, Isolation, In Vitro, Formalin-fixed Paraffin-Embedded, Staining