human cd63 mouse dshb h5c6  (Developmental Studies Hybridoma Bank)

Journal: iScience

Article Title: Plasma extracellular vesicle modulate immune cell transcriptional responses following acute myocardial infarction

doi: 10.1016/j.isci.2026.114665

Figure Lengend Snippet: Characterization of plasma EV in MI patients EVs were isolated from the plasma of MI patients at hospital presentation and at 3-month follow-up. (A) EV-Array analysis of canonical EV-associated markers shows significant enrichment at MI presentation ( n = 17 patients paired at both timepoints). (B) EV-cell-associated markers were also enriched at presentation with MI. White in the heatmaps denotes the baseline (normalized to 1), corresponding to the 3-month follow-up value. (C) Total EV concentrations measured by NTA of SEC and ultracentrifugation-purified plasma EVs. (D and E) Spaghetti plots showing EV size and particle concentration profiles at the individual patient level across the two timepoints ( n = 19 patients paired at both timepoints). (E) Western blotting confirmed the presence of EV markers (CD9 and Syntenin-1), absence of cellular contaminants (GM130, ATP5A, and Histone H3), and depletion of the plasma Apo B. (F) TEM images confirmed typical cup-shaped EV morphology. Scale bars, 500 nm or 100 nm. (G and H) Single-particle interferometric reflectance imaging (NanoView) confirmed tetraspanin-positive EVs (CD9, CD63, and CD81), (H) with associated size and concentration profiles ( n = 9–10 at both timepoints). EV marker values at MI presentation were normalized to each patient’s own 3-month follow-up value. In (G), data are presented as group means ± SD. Statistical analysis: (A–C) Wilcoxon matched-pairs signed-rank test; (G) two-way ANOVA with Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: CD63 , Biorad , MCA2142; RRID: AB_324562.

Techniques: Clinical Proteomics, Isolation, Purification, Concentration Assay, Western Blot, Single Particle, Imaging, Marker

Journal: iScience

Article Title: Plasma extracellular vesicle modulate immune cell transcriptional responses following acute myocardial infarction

doi: 10.1016/j.isci.2026.114665

Figure Lengend Snippet: Enrichment of miRNA-320b in endothelial cell-derived EV following inflammatory stimulation HUVECs were cultured under control (black) or TNF-α-stimulated (red) conditions to model endothelial inflammation. (A) Total and viable cell counts at study endpoint confirm similar cell numbers across conditions. (B) Soluble VCAM-1 levels in supernatants were significantly increased following TNF-α stimulation, confirming proinflammatory activation. (C and D) Single-particle interferometric reflectance imaging (NanoView) detected CD9, CD63, and CD81-positive EVs in both conditions. Scale bars, 10 μm. (D) Total captured EV concentrations were significantly higher in TNF-α-stimulated cultures. (E) Average particle diameters of captured EVs did not differ significantly. (F) NTA showed elevated particle concentrations in EV preparations from TNF-α-treated HUVECs. (G) Size distribution profiles of EVs from control and TNF-α conditions. (H) EV counts were normalized to total and live cell numbers, confirming increased release under inflammatory stimulation. (I) Western blot analysis of HUVEC-derived EVs confirmed enrichment of canonical EV markers (TSG101, Syntenin-1, and CD9), absence of cellular contaminant GM130, and presence of VCAM-1 specifically in TNF-α-derived EVs. (J) TEM images showed characteristic EV morphology; scale bars, 1 μm or 500 nm. (K) RT-qPCR showed no change in HUVEC cellular miRNA-320b levels between conditions. (L) Significant enrichment of miRNA-320b was detected in EVs from TNF-α-stimulated HUVECs. miRNA expression was normalized to UniSp6 spike-in control, because endogenous controls (e.g., miR-103a-3p) were undetectable in EV samples. Data are presented as group means ± SD. N = 4 biological replicates per group. Statistical analysis: (B, F, L) unpaired Student’s t test; (D, H) two-way ANOVA with Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: CD63 , Biorad , MCA2142; RRID: AB_324562.

Techniques: Derivative Assay, Cell Culture, Control, Activation Assay, Single Particle, Imaging, Western Blot, Quantitative RT-PCR, Expressing

Journal: Cell reports

Article Title: Transplants foster B cell alloimmunity by relaying extracellular vesicles to follicular dendritic cells

doi: 10.1016/j.celrep.2025.115832

Figure Lengend Snippet: (A) Donor H2 Ag in splenic FDCs (inset, arrows) after transplantation of B6 (H2 b ) hearts in BALB/c mice. Original magnification ×200. Scale bars, 20 μm. (B) Quantification with ImageJ of donor H2 Ag spots per FDC network on spleens of BALB/c mice transplanted with B6 hearts. (C) Donor H2 Ag spots per FDC network on BALB/c LNs draining B6 skin allografts assessed using ImageJ. (D) Deep SIM of donor H2 Ag spots in FDCs of a BALB/c LN draining a B6 skin graft. Scale bar, 2 μm. ROI, region of interest. (E and F) 2P-microscopy of FDCs in the spleen of a BALB/c mouse transplanted with a B6 heart releasing RFP-sEVs (E) and a BALB/c LN draining a B6 skin graft releasing RFP-sEVs (F). Arrows: graft-RFP-sEVs on the surface (red) or inside (yellow) of FDCs (green). CellTrace Violet-B cells indicate B cell follicles. FDCs were labeled with AF488-CD21/CD35 Ab. Original magnification ×25. Scale bars, 20 μm. (G) Colocalization by confocal microscopy of donor (B6, IA b + H2K b ) allo-Ags in FDCs of LNs draining CMV Cre/+ LSL -RFP-CD63 B6 skin grafted in BALB/c mice. Scale bar, 10 μm. Mander’s colocalization coefficient of graft-sEVs and donor IA b + H2K b Ab analyzed using Imaris. Each triangle represents one FDC network. In (E) and (F), RFP spots per FDC network z stacks were quantified within 90-μm-thick z stacks using Imaris. Experiments were done on 4 spleens per POD (A and B), 6 graft-dLNs per POD (C), 3 graft-dLNs (D), 4 spleens (E), 4 graft-dLNs (F), and 12 graft-dLNs (G). In (B), (C), (E), and (F), comparisons were made by multiple unpaired two-tailed Student’s test. Error bars, means ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; NS, not significant.

Article Snippet: Anti-human / mouse CD63 (Clone LAMP3/ 2788) , NeoBiotechnologies , Cat # 967-MSM8-P1.

Techniques: Transplantation Assay, Microscopy, Labeling, Confocal Microscopy, Two Tailed Test

Journal: Cell reports

Article Title: Transplants foster B cell alloimmunity by relaying extracellular vesicles to follicular dendritic cells

doi: 10.1016/j.celrep.2025.115832

Figure Lengend Snippet: (A) FACS analysis of B6 LN FDC-enriched suspensions incubated with CM-DiI-allo (BALB/c)-sEVs. FDCs with remnant B cells attached were excluded by gating CD19 Neg FDCs. One of two experiments. (B) Three-dimensional reconstruction by STED microscopy of a B6 FDC following incubation with CM-DiI-allo (BALB/c)-sEVs from the experiment in (A). Top: sEVs bound to the FDC. Bottom: 180° rotation to visualize internalized sEVs at the FDC midsection. Scale bars, 5 μm. (C) IEM of an FDC (pseudo-colored) in the dLN of a BALB/c mouse injected in the footpad with 10-nm-gold CD21/35 Ab to label FDCs and allo (B6)-sEVs coated with 5-nm-gold H2K b -IA b Abs. Insets: allo-sEVs next to an FDC. N, nucleus; ROI, region of interest. Scale bars, 200 nm (D) IEM of allo (B6)-sEVs in endocytic vesicles of a BALB/c LN FDC, 3 h after footpad injection of the sEVs. Scale bars, 100 nm (E) IEM of footpad-injected allo (B6)-sEVs (5 nm gold) internalized into the labyrinthine infoldings of FDCs heavily labeled with 10-nm-gold CD21/CD35 Ab. N, nucleus. Scale bar, 300 nm. (F) Diagram of pHluorin-CD63-mScarlet-tagged sEVs internalized by FDCs in dLNs and then recycled to the extracellular space, where the sEVs emit green flashes when exposed to neutral pH. Illustration created with BioRender. (G) IEM of pHluorin labeled with 6 nm gold (arrows) on the B6 sEV surface. Scale bar, 50 nm. (H) Time-lapse by 2P-microscopy on an explanted BALB/c LN of recycling of pHluorin-CD63-mScarlet-tagged B6 sEVs from intracellular compartments of FDCs (arrows at 0 and 45 seconds) to the surface of the FDC and extracellular milieu (arrow at 90 seconds), where the sEVs emitted green flashes. Scale bar, 50 μm. For (C)–(E) and (G), original magnifications were ×20,000 and ×80,000.

Article Snippet: Anti-human / mouse CD63 (Clone LAMP3/ 2788) , NeoBiotechnologies , Cat # 967-MSM8-P1.

Techniques: Incubation, Microscopy, Injection, Labeling

Journal: Cell reports

Article Title: Transplants foster B cell alloimmunity by relaying extracellular vesicles to follicular dendritic cells

doi: 10.1016/j.celrep.2025.115832

Figure Lengend Snippet: (A) Isolation of sEVs from culture supernatants of WT or Rab27a KO B6 mouse skin explants under pro-inflammatory conditions. Diagram created with BioRender. (B) Microscopy of cryosections of BALB/c LNs draining WT or Rab27a KO B6 skin allografts. Donor H2 Ag was detected using a cocktail of biotin-IA b and -H2K b Abs. Original magnification ×200. Representative of eight LNs per variable. Scale bars, 30 μm. (C) Quantification with ImageJ of donor (B6) H2 Ag Pos spots on FDCs in skin graft-dLNs pooled from eight BALB/c recipients per POD (left). DSAs (FACS) in sera of BALB/c mice transplanted with WT or Rab27a KO B6 skin (right). Each symbol represents one recipient. (D) EM of CM-DiI-labeled sEVs from supernatants of human DCs. Original magnification ×80,000. Scale bar, 200 nm. (E) Western blot with sEV markers Tsg101 and CD63, and lack of the endoplasmic reticulum marker GRP94, on the human sEVs used in the experiments in (F)–(I). (F) Quantification (Imaris) of CM-Dil Pos spots in FDCs on cryosections of human spleens incubated with CM-DiI-human allo-sEVs untreated or opsonized with normal human serum, unprocessed or heat de-complemented. (G) Representative cryosections of human spleen incubated (or not, control) with CM-DiI-human allo-sEVs (red) untreated or opsonized with normal or heat-decomplemented human serum. FDCs are labeled with CD35 Ab (green). Arrows indicate CM-DiI-human allo-sEVs retained on the tissue cryosections. Original magnification ×400. Scale bar, 10 μm. (H) Overlapping of CM-DiI-human allo-sEVs and human splenic FDCs. (I) STED microscopy revealed that the CM-Dil Pos spots (circles) by fluorescence microscopy in human FDCs are clusters of sEVs. Scale bars, 0.5 μm. In (C) and (F), comparisons were by two-tailed Student’s test. Error bars, means ± SD; ** p < 0.01, *** p < 0.001, and **** p < 0.0001; NS, not significant.

Article Snippet: Anti-human / mouse CD63 (Clone LAMP3/ 2788) , NeoBiotechnologies , Cat # 967-MSM8-P1.

Techniques: Isolation, Microscopy, Labeling, Western Blot, Marker, Incubation, Control, Fluorescence, Two Tailed Test