h5c6 rrid ab 2884028 cd81 1d6 mouse mab novus biologicals cat nb100 65805  (Novus Biologicals)

Journal: Bioactive Materials

Article Title: ADGRG1-targeted hypoxia preconditioned extracellular vesicles ameliorate intervertebral disc degeneration by delivering taurine to disrupt the oxidative stress feedback loop-driven ferroptosis in nucleus pulposus cells

doi: 10.1016/j.bioactmat.2026.02.029

Figure Lengend Snippet: Preparation and characterization of engineered ADGRG1-targeting and hypoxia-treated EVs. (A)Induced fit docking analysis of ADGRG1-binding peptide (A1TP, 7 peptides) and extracellular domain of ADGRG1 protein (PDB database: 7SF8). (B) Analysis of the binding of the A1TP to purified ADGRG1 proteins using a microscale thermophoresis (MST) binding assay. (C) Induced fit docking analysis of A1TP-PEG and extracellular domain of ADGRG1 protein. (D) The binding free energy between A1TP or A1TP-PEG and ADGRG1 were calculated using molecular dynamics simulations. Lower values indicate more stable interactions, with values less than or equal to −20 considered as stable binding modes. (E) Schematic illustration of the conjugating reaction between DSPE-PEG-Alkyne and A1TP. Schematic illustration of the fabrication of A1TP-HX-EVs through external modification by A1TP anchoring. Specific steps for the synthesis of DSPE-PEG-A1TP (DPA) are shown in . (F) FT-IR analysis showed the characteristic peaks of the DSPE-PEG-A1TP. The new triazole ring itself showed a characteristic C=N stretching vibration, a peak at 1538 cm −1 revealed the successful conjugation of A1TP. (G) H Nuclear magnetic resonance (NMR) spectra of DSPE-PEG-A1TP in D2O. The hydrogen signatures of the phenyl and phenol groups at 7.5-8.0 ppm confirmed the successful conjugation of DSPE to A1TP. (H) Western blot analysis verified the presence of three EV marker proteins (ALIX, TSG101, and CD81) and one EV negative marker (GM130) in EVs, HX-EVs, and A1TP-HX-EVs. (I) Transmission electron microscopy (TEM) images of EVs, HX-EVs and A1TP-HX-EVs. Scale bar, 200 nm. (J) Zeta potentials of EVs, HX-EVs and A1TP-HX-EVs, n = 3. Two-tailed unpaired Student's t-test was used for statistical analysis. ns, not significant. A two-tailed unpaired Student's t-test was used for statistical analysis. (K) Representative images of the spherical morphology and dispersion states of EVs, HX-EVs and A1TP-HX-EVs. Scale bar, 500 nm. (L) Size distributions of EVs, HX-EVs and A1TP-HX-EVs.

Article Snippet: Finally, the presence of the characteristic EV markers Alix (92880, Cell Signaling Technology), CD81 (56039, Cell Signaling Technology) and TSG101 (sc-7964, Santa Cruz Biotechnology) was confirmed by Western blot analysis.

Techniques: Binding Assay, Purification, Microscale Thermophoresis, Modification, Conjugation Assay, Nuclear Magnetic Resonance, Western Blot, Marker, Transmission Assay, Electron Microscopy, Two Tailed Test, Dispersion

Journal: Bioactive Materials

Article Title: NsPEFs-enriched ADSCs-EVs alleviate osteoarthritis via RSPO3-mediated dual pro-chondrogenic and pro-M2 macrophage properties

doi: 10.1016/j.bioactmat.2026.01.006

Figure Lengend Snippet: NsPEFs engineering boosts the production of ADSCs-EVs with superior yield and stability A. Schematic illustration of the high-efficiency extraction of extracellular vesicles (EVs) from adipose-derived stem cells (ADSCs) using nanosecond pulsed electric fields (NsPEFs). B. Representative transmission electron microscopy (TEM) images of isolated Ctrl-ADSCs-EVs and NsPEFs-ADSCs-EVs, showing characteristic cup-shaped morphology and bilayer membrane (scale bars: 150 nm and 75 nm). C. Nanoparticle tracking analysis (NTA) showing the particle size distribution of EVs (n = 3). D. Western blot (WB) analysis confirming the positive expression of EV-specific markers (CD81, CD63, TSG101) and the absence of the negative markers (Calnexin, Histone H3, LaminA/C). Quantification is shown on the right (n = 3). E. The particle concentration of EVs. F. NsPEFs stimulation significantly enhanced both yield and protein output compared to Ctrl-ADSCs-EVs. G. Zeta potential measurement indicating colloidal stability (n = 3). H. Purity assessment expressed as the particle-to-protein ratio ( × 10 9 particles/μg). I. Viability of cells post-NsPEFs-ADSCs-EVs treatment assessed by trypan blue exclusion assay (scale bar: 1.7 mm). Data are presented as mean ± SEM from at least three independent experiments. Statistical significance was determined by unpaired two-tailed Student's t-test or one-way ANOVA with Tukey's post-hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001; ns: not significant.

Article Snippet: The antibodies used and the dilution ratios were as follows:Anti-INOS (1:800, Cohesion), Anti-Arginase 1 (1:800, BOSTER), Anti-LRP6 (1:800, BOSTER), Anti-Beta-catenin (1:800, BOSTER), Anti-CD163 (1:800, Abclonal), Anti-CD86 (1:800, BOSTER), Anti-LGR4 (1:800, Abclonal), Anti-IL-1β (1:800, BOSTER), Anti-IL-10 (1:1000, Bioss), Anti-MMP13 (1:800, BOSTER), Anti-COL2A1 (1:800, BOSTER), Anti-Histone H3 (1:1000, Nature Biosciences), Anti-Lamin A/C (1:1000, Nature Biosciences), Anti-Akt (1:1000, Nature Biosciences), Anti-pAkt (1:1000, Nature Biosciences), Anti-RSPO3 (1:1000, Abcam), Anti-CD63(1:800, BOSTER), Anti-CD81(1:800, BOSTER), Anti-TSG101(1:800, BOSTER), Anti-Calnexin(1:800, BOSTER).

Techniques: Extraction, Derivative Assay, Transmission Assay, Electron Microscopy, Isolation, Membrane, Western Blot, Expressing, Concentration Assay, Zeta Potential Analyzer, Trypan Blue Exclusion Assay, Two Tailed Test

Journal: Neural Regeneration Research

Article Title: R-28 cell-derived extracellular vesicles protect retinal ganglion cells in glaucoma

doi: 10.4103/NRR.NRR-D-24-00709

Figure Lengend Snippet: The diameter and concentration of particles present in EV preparation were determined by nanoparticle analysis and immunoblotting with EV-specific markers. EV concentration by particle diameter was obtained from Nanosight (A) after isolation from R-28 cells shown as magnified at 10×, Scale bar: 50 µm (B). EVs were enriched for the EV marker CD81 compared to R-28 cell lysates when the same density of protein was loaded (C). Over three separate isolations, the average concentration (D) and size of the EVs (E) were determined by Nanosight ( n = 3). Electron microscopic imaging of the R-28 cells with EVs on the cell surface is visible (inset; F), and EVs were also detected in the purified EV preparation (G). EV: Extracellular vesicle.

Article Snippet: CD81 , Hamster , BioRad, Watford, UK , 1:100 , MCA1846 , N/A.

Techniques: Concentration Assay, Western Blot, Isolation, Marker, Imaging, Purification