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

Journal: Journal of Extracellular Vesicles

Article Title: Nano‐Flow Cytometry‐Guided Discrimination and Separation of Human Cytomegalovirus Virions and Extracellular Vesicles

doi: 10.1002/jev2.70060

Figure Lengend Snippet: Size exclusion chromatography purification of EVs from post‐25,000 × g centrifugation supernatants. EVs remaining in the supernatants following high‐speed centrifugation were concentrated by 100‐kDa ultrafiltration, and fractionated on a custom Sepharose CL‐2B column. (A) EV/protein elution profiles as measured by nanoparticle tracking analysis (NTA) and Bradford assay show a robust separation of EVs from the contaminating protein. (B) Silver staining of fractions 6–35 from the HCMV‐infected cells, including the starting conditioned medium (CCM), and the non‐conditioned medium (NCM). Bovine serum albumin (BSA), the most abundant component of the fetal bovine serum (FBS) in the complete growth medium, appeared in fraction 19 and peaked in fraction 31 (indicated with red rectangles). (C) ELISA for CD63 and β‐actin shows positivity for these two EV‐associated proteins in the particle‐containing fractions. Matching NTA and Bradford assay data obtained from non‐conditioned complete medium is shown in Figure while the matching silver staining data for uninfected cells is shown in Figure . Data in panel A are presented as means ± SEMs ( N = 3), with all measurements performed in triplicate. Data included in panels B and C are from single experiments. ELISA measurements were performed in duplicate. F = fraction; M = molecular weight markers.

Article Snippet: Blocking was performed with 1% bovine serum albumin (BSA; from Sigma‐Aldrich) in DPBS for 1 h. Primary antibodies raised against CD63 (clone MEM‐259; BioRad item MCA2142) or beta‐actin (clone AC‐74; Sigma‐Aldrich item A2228‐100UL) were diluted in blocking buffer to 1 and 2 μg/mL, respectively, and added overnight at 4°C.

Techniques: Size-exclusion Chromatography, Purification, Centrifugation, Bradford Assay, Silver Staining, Infection, Enzyme-linked Immunosorbent Assay, Molecular Weight

Journal: STAR Protocols

Article Title: Protocol for the isolation and characterization of murine hematopoietic stem and progenitor cell-derived extracellular vesicles

doi: 10.1016/j.xpro.2025.103778

Figure Lengend Snippet: Representative eSRRF graph to analysis EVs markers Area, circularity and positivity for the CD63 marker in HPSC-derived EVs using eSRRF technique compared to beads and PBS as positive and negative controls, respectively; Images adapted from Bonora et al., 2024.

Article Snippet: PE mouse anti-human CD63 (dilution 1:100) , eBioscience , Cat# 12-0639-42; RRID: AB_2572565.

Techniques: Marker, Derivative Assay

Journal: STAR Protocols

Article Title: Protocol for the isolation and characterization of murine hematopoietic stem and progenitor cell-derived extracellular vesicles

doi: 10.1016/j.xpro.2025.103778

Figure Lengend Snippet:

Article Snippet: PE mouse anti-human CD63 (dilution 1:100) , eBioscience , Cat# 12-0639-42; RRID: AB_2572565.

Techniques: Magnetic Beads, Recombinant, Control, Isolation, Software

Journal: Bioactive Materials

Article Title: Mesenchymal stromal/stem cell spheroid-derived extracellular vesicles advance the therapeutic efficacy of 3D-printed vascularized artificial liver lobules in liver failure treatment

doi: 10.1016/j.bioactmat.2025.02.042

Figure Lengend Snippet: Characterization of extracellular vesicles (EVs) . (a) A schematic illustrates the extraction procedures of EVs from MSCs either cultured as dissociated single cells on 2D tissue culture plates (SiEV) or as cell spheroids (SpEV). The optical microscopy image shows the MSC spheroids produced on the microwells. Scale bar: 200 μm. (b) The TEM images of SiEV and SpEV. Scale bar: 100 nm. (c) The particle concentration and median diameter of SiEV and SpEV measured by nanoparticle tracking analysis (NTA). Data are presented as mean ± SEM, n = 3. (d) Western blots of the EV surface markers (CD9, CD63, CD81) and the reference GAPDH. (e–f) Pearson's correlation analysis and principal component analysis (PCA) of the gene expression among different samples. (g) The heat maps of partially differently expressed cytoplasmic and extracellular proteins in the extracellular vesicles, p < 0.05, fold change (FC) > 1.2. (h) The interaction network of differently expressed plasma membrane proteins in SiEV and SpEV. The line thickness reflects the strength of The levels of hub proteins (STON2, NOTCH1, and DAAM1) in SiEV and SpEV. All data are normalized to the value of the “SiEV” group and presented as mean ± SEM, n = 2. Significance levels: 0.01 < ∗ p < 0.05, ∗∗ p < 0.01, not significant (ns) for p > 0.05. (j) The gene set enrichment analysis (GSEA) plot highlights the elevated NOTCH signaling in SpEV according to the Reactome database, p < 0.05, normalized enrichment score (NES) > 1. (k) The gene ontology (GO) analysis of partial differently upregulated biological processes in SpEV, p < 0.05, false discovery rate (FDR) < 0.1.

Article Snippet: The primary antibodies, including mouse-anti-human CD63 (1 : 5000 dilution, Proteintech, China), mouse-anti-human CD9 (1 : 5000 dilution, Proteintech, China), rabbit-anti-human CD81 (1 : 1000 dilution, Abcam, USA), as well as the HRP-conjugated goat-anti-mouse secondary antibody (1 : 5000 dilution, Beyotime, China) and the HRP-conjugated goat-anti-rabbit secondary antibody (1 : 5000 dilution, Proteintech, China) were used to identify the typical markers of extracellular vesicles.

Techniques: Extraction, Cell Culture, Microscopy, Produced, Concentration Assay, Western Blot, Gene Expression, Clinical Proteomics, Membrane

Journal: Molecular Medicine

Article Title: Extracellular vesicles from microglial cells activated by abnormal heparan sulfate oligosaccharides from Sanfilippo patients impair neuronal dendritic arborization

doi: 10.1186/s10020-024-00953-1

Figure Lengend Snippet: Western blot antibodies

Article Snippet: CD63 , Mouse anti-mouse, rat, human , 1:500 , Santa Cruz (Sc-5275).

Techniques: Western Blot

Journal: bioRxiv

Article Title: The First Comprehensive Description of the Platelet Single Cell Transcriptome

doi: 10.1101/2024.10.15.618506

Figure Lengend Snippet: A) Total mRNA molecules per cell for the four healthy donors evaluated by Abseq censored based on the presence of CD41 oligo, B) UMAP of the four healthy donors coded by color. C) Hi stringency UMAP (D) and low stringency UMAP of the four healthy donors. E) Correlation of CD41 protein expression with ITGA2B mRNA expression. F) Correlation of CD63 protein expression with CD63 mRNA within merged dataset. G) Bar plot of the cell level % expression of mRNA for CD63 and ITGA2B relative to protein levels.

Article Snippet: The Ab-Oligos mouse anti-human CD41a, mouse anti-human CD62P, and mouse anti-human CD63 were used to stain samples for further proteomic labeling.

Techniques: Expressing