Journal: International Journal of Molecular Sciences

Article Title: Effect of Small Extracellular Vesicles Produced by Mesenchymal Stem Cells on 5xFAD Mice Hippocampal Cultures

doi: 10.3390/ijms26094026

Figure Lengend Snippet: Characteristics of sEVs purified by asymmetric depth filtration. ( a ): Heterogeneity of sEVs as determined by TEM. ( a – c ): Protein content of MSC supernatant (MSC SN), and the flow through after sEV purification (Flow) and in sEVs found by 41-plex assay, respectively. Frac denotes fractalkine. A total of 6 flow-through, 12 MSC SN, and 6 sEV samples were used (see for details). ( d , e ): NTA analysis of the hydrodynamic diameter of sEVs (pdf—probability density function) ( d ) and confocal image ( e ) of sEVs stained with lipid dye BDP (red). ( f ): Western blot and 12% PAAG of sEVs and control cells (A431) stained with antibodies to calnexin, CD9, and HSP70 (M—protein ladder).

Article Snippet: The proteins were then transferred to PVDF membranes (BioRad Laboratories, Hercules, CA, USA), and 5% skimmed milk was used to block the membranes at room temperature for 1 h. Subsequently, the membranes were incubated with primary antibodies to CD9, HSP70, or calnexin (AffiGEN, San Jose, CA, USA) at 4 °C overnight, washed out, and secondary anti-rabbit HRP or anti-mouse HRP antibodies (RRID: AB_2650737 and AB_315009, Biolegend, San Diego, CA, USA) were added at room temperature for 1 h. Protein bands were visualized using 3,3′-Diaminobenzidine tetrahydrochloride (DAB, Merck KGaA, Darmstadt, Germany) and a gel imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Techniques: Purification, Filtration, Plex Assay, Staining, Western Blot, Control