Journal: The Journal of Cell Biology

Article Title: A novel role for FGF and extracellular signal–regulated kinase in gap junction–mediated intercellular communication in the lens

doi: 10.1083/jcb.200101057

Figure Lengend Snippet: Vitreous humor contains a gap junctional intercellular communication-promoting activity with properties of an FGF. 50 ng/ml FGF-2 in M199/BOTS or E10 chick vitreous humor diluted with 2.3 vol of M199/BOTS (30% VH) was incubated with heparin-conjugated Affigel beads in the presence of 0.1 M NaCl or 0.6 M NaCl as described in Materials and methods. The beads were pelleted, and after removal of the supernatant (heparin 0.1 M or 0.6 M NaCl unbound), FGF-like activity was eluted with 2.5 M NaCl (heparin 2.5 M NaCl eluate). The fractions were then brought to 0.15 M NaCl by repeated rounds of concentration and dilution with M199 medium. DCDMLs were cultured for 2 d in M199/BOTS with no additions (control), in M199/BOTS with unfractionated FGF-2 (50 ng/ml FGF-2 input), in unfractionated 30% VH (30% VH input), or with the indicated heparin-Affigel fraction. Gap junctional intercellular communication was assessed as described in the legend to A; only Lucifer yellow immunofluorescence is presented. In all cases, rhodamine-dextran was confined to a single row of cells immediately bordering the wound. Representative results from three independent experiments; similar results were obtained with vitreous body conditioned medium (not shown). Note that the heparin beads quantitatively bound the communication-promoting activity of even the highest concentration of FGF tolerated by lens cells (50 ng/ml). The values given underneath the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untreated controls within the same experiment. Only the values obtained for the 30% VH input and the heparin 2.5 M NaCl eluate were significantly higher than control ( P < 0.03). Bar, 50 μm.

Article Snippet: 1 ml of either vitreous humor diluted with 2.3 vol of M199 (30% VH/M199) or M199 containing 50 ng/ml of FGF-2 was mixed end-over-end with 0.1 ml of heparin-conjugated Affigel beads (Bio-Rad Laboratories) in the presence of either 0.1 M NaCl or 0.6 M NaCl for 2 h at 4°C.

Techniques: Activity Assay, Incubation, Concentration Assay, Cell Culture, Control, Immunofluorescence, Standard Deviation

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