Journal: International Journal of Molecular Sciences

Article Title: Analysis of Proteins and Peptides of Highly Purified CD9 + and CD63 + Horse Milk Exosomes Isolated by Affinity Chromatography

doi: 10.3390/ijms232416106

Figure Lengend Snippet: Affinity chromatography of VESmix on anti-CD9- ( A ) and anti-CD63-Sepharose ( B ). ( – )—absorbance on A 280 nm, mAU; P1—subfraction appeared in flow, P2—subfraction eluted with 0.5 NaCl, P3—subfraction eluted with 1.0 M NaCl, P4—subfraction eluted with 0.1 M Gly-HCl pH 2.6.

Article Snippet: Exosome preparations were applied to anti-CD9- and anti-CD63-Sepharoses equilibrated with 20 mM Tris-HCl pH 7.5; the sorbents were washed with the same buffer to zero A 280 density (peak P1).

Techniques: Affinity Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Proteins and Peptides of Highly Purified CD9 + and CD63 + Horse Milk Exosomes Isolated by Affinity Chromatography

doi: 10.3390/ijms232416106

Figure Lengend Snippet: Transmission electron microscopy of horse milk exosomes on different stages of isolation and purification. P2 subfraction was obtained after affinity chromatography on anti-CD9-Sehparose ( A – D , I ) and anti-CD63-Sepharose ( E – H , J ). General view ( A , E ); exosomes of ≤ 100 nm ( B , F ); vesicles > 100 nm ( C ); exosomes (white arrow) and particles of medium electron density without outer membranes (ovals) ( D ); particles of medium electron density without outer membranes ( G ); vesicles, “non-vesicles”, particles of medium electron density without outer membranes ( H ); gold immunolabeling using anti-CD9-IgG ( I ) and anti-CD63-IgG ( J ). The length of the scale line corresponds to 100 nm ( B , D , F – J ) and 200 nm ( A , C , E ).

Article Snippet: Exosome preparations were applied to anti-CD9- and anti-CD63-Sepharoses equilibrated with 20 mM Tris-HCl pH 7.5; the sorbents were washed with the same buffer to zero A 280 density (peak P1).

Techniques: Transmission Assay, Electron Microscopy, Isolation, Purification, Affinity Chromatography, Immunolabeling

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Proteins and Peptides of Highly Purified CD9 + and CD63 + Horse Milk Exosomes Isolated by Affinity Chromatography

doi: 10.3390/ijms232416106

Figure Lengend Snippet: Flow cytometry analysis of P2 + P3 subfractions eluted from anti-CD9- and anti-CD63-Sepharose. Relative amounts of exosomes containing CD9 (left panel), CD63 (central panel), and CD81 (right panel) are shown.

Article Snippet: Exosome preparations were applied to anti-CD9- and anti-CD63-Sepharoses equilibrated with 20 mM Tris-HCl pH 7.5; the sorbents were washed with the same buffer to zero A 280 density (peak P1).

Techniques: Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Proteins and Peptides of Highly Purified CD9 + and CD63 + Horse Milk Exosomes Isolated by Affinity Chromatography

doi: 10.3390/ijms232416106

Figure Lengend Snippet: Direct MALDI mass spectrometry analysis of untreated exosomes (P2 + P3) after affinity chromatography on anti-CD9- ( A ) and anti-CD63-Sepharose ( B ). The errors in determining the m / z value did not exceed 0.5–1.0 Da.

Article Snippet: Exosome preparations were applied to anti-CD9- and anti-CD63-Sepharoses equilibrated with 20 mM Tris-HCl pH 7.5; the sorbents were washed with the same buffer to zero A 280 density (peak P1).

Techniques: Mass Spectrometry, Affinity Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Proteins and Peptides of Highly Purified CD9 + and CD63 + Horse Milk Exosomes Isolated by Affinity Chromatography

doi: 10.3390/ijms232416106

Figure Lengend Snippet: MALDI mass spectrometry analysis of P2 + P3 exosome extracts obtained after affinity chromatography on anti-CD9- ( A ) and anti-CD63-Sepharose ( B ). The extracts were obtained after the destruction with TFA and acetonitrile. The error in determining the m / z values did not exceed 0.5–1.0 Da.

Article Snippet: Exosome preparations were applied to anti-CD9- and anti-CD63-Sepharoses equilibrated with 20 mM Tris-HCl pH 7.5; the sorbents were washed with the same buffer to zero A 280 density (peak P1).

Techniques: Mass Spectrometry, Affinity Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Proteins and Peptides of Highly Purified CD9 + and CD63 + Horse Milk Exosomes Isolated by Affinity Chromatography

doi: 10.3390/ijms232416106

Figure Lengend Snippet: MALDI mass spectra of P2 + P3 subfraction of horse milk exosome extracts obtained after affinity chromatography on anti-CD9- ( A ) and anti-CD63-Sepharose ( B ). The extracts were obtained by destruction of P2 subfraction with TFA and acetonitrile, and the compounds passed through the Amicon filter with a 3 kDa cut-off were analyzed. The error in determining the m / z values did not exceed 0.5–1.0 Da.

Article Snippet: Exosome preparations were applied to anti-CD9- and anti-CD63-Sepharoses equilibrated with 20 mM Tris-HCl pH 7.5; the sorbents were washed with the same buffer to zero A 280 density (peak P1).

Techniques: Affinity Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Proteins and Peptides of Highly Purified CD9 + and CD63 + Horse Milk Exosomes Isolated by Affinity Chromatography

doi: 10.3390/ijms232416106

Figure Lengend Snippet: MALDI mass spectra of horse milk exosome extract corresponding to peak P2 after affinity chromatography on anti-CD9-Sepharose before ( A ) and after proteolysis with trypsin ( B ), chymotrypsin ( C ), and proteinase K ( D ). The error in determining the m / z value did not exceed 0.5–1.0 Da.

Article Snippet: Exosome preparations were applied to anti-CD9- and anti-CD63-Sepharoses equilibrated with 20 mM Tris-HCl pH 7.5; the sorbents were washed with the same buffer to zero A 280 density (peak P1).

Techniques: Affinity Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Proteins and Peptides of Highly Purified CD9 + and CD63 + Horse Milk Exosomes Isolated by Affinity Chromatography

doi: 10.3390/ijms232416106

Figure Lengend Snippet: Molecular masses of major peptides corresponding to P2 + P3 peaks of VESmix isolated by affinity chromatography on anti-CD9/CD63-Sepharoses and extracts obtained using TFA and acetonitrile.

Article Snippet: Exosome preparations were applied to anti-CD9- and anti-CD63-Sepharoses equilibrated with 20 mM Tris-HCl pH 7.5; the sorbents were washed with the same buffer to zero A 280 density (peak P1).

Techniques: Isolation, Affinity Chromatography

Journal: Cells

Article Title: Beneficial Effect of ACI-24 Vaccination on Aβ Plaque Pathology and Microglial Phenotypes in an Amyloidosis Mouse Model

doi: 10.3390/cells12010079

Figure Lengend Snippet: ACI-24 vaccination efficiently reduces Aβ plaque load in APPPS1 mice. Schematic overview of the vaccination paradigm ( A ). ELISA analysis of the anti-Aβ42 total IgG titres (ng/mL) in plasma ( B ). Control APPPS1 mice (PBS treated, in dotted grey) have the baseline titres, while both ACI-24 vaccinated APPPS1 (in red) and ACI-24 vaccinated WT mice (in black) have robust and sustained immune response. Representative images of Aβ (as stained with NAB228 antibody) and nuclei (Hoechst) in cortical sections show decreased Aβ deposition following ACI-24 vaccination. Scale bar for images in the centre column is indicating 200 µm, and scale bar for images in the right column (magnification of dotted boxed regions from centre column images) is indicating 50 µm ( C ). Statistical analysis of 10 control and 10 ACI-24 vaccinated APPPS1 mice for Aβ plaque coverage ( D ), number ( E ) and size ( F ). ACI-24 significantly downregulated Aβ plaque coverage and size. Offspring are from the transgenic mother (pink circles) or father (blue circles). Graphs are presented as mean ± SEM (* p < 0.05, ** p < 0.001, unpaired two-tailed Student’s t -test).

Article Snippet: Primary antibodies used: Aβ 3552 (0.74 μg/mL) [ ], Aβ NAB228 (1:500, sc-32277, Santa Cruz Biotechnology, Heidelberg, Germany) [ ], BACE1 (1:400, ab108394, Abcam, Cambridge, UK), ApoE (1:200, hybridoma supernatant, clone 26C11), IBA1 (1:500, ab5076, Abcam, Cambridge, UK), IBA1 (1:500, 234308, Synaptic Systems, Göttingen, Germany), NLRP3 (1:50, BSS-BS-10021R-100, Biozol Diagnostics Vertrieb, Eching, Germany) and CD68 (1:500, MCA1957GA, Bio-Rad, Feldkirchen, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Control, Staining, Transgenic Assay, Two Tailed Test

Journal: Cells

Article Title: Beneficial Effect of ACI-24 Vaccination on Aβ Plaque Pathology and Microglial Phenotypes in an Amyloidosis Mouse Model

doi: 10.3390/cells12010079

Figure Lengend Snippet: ACI-24 reduces ApoE protein levels and neuronal injury in the offspring generated by the transgenic mother. Representative images of control and ACI-24 vaccinated mice stained for nuclei (Hoechst), fibrillar Aβ (ThR), Aβ plaques (3552 antibody) and ApoE ( A ); and nuclei (Hoechst), fibrillar Aβ (ThR), Aβ plaques (NAB228 antibody) and BACE1 (dystrophic neurites) ( B ). Scale bar: 200 µm. Statistical analysis of 5 control and 6 ACI-24 vaccinated mice (transgenic mother offspring) showing significant downregulation of both total ApoE ( C ) and BACE1 ( D ) coverage. Graphs are presented as mean ± SEM (* p < 0.05, ** p < 0.001, unpaired two-tailed Student’s t -test).

Article Snippet: Primary antibodies used: Aβ 3552 (0.74 μg/mL) [ ], Aβ NAB228 (1:500, sc-32277, Santa Cruz Biotechnology, Heidelberg, Germany) [ ], BACE1 (1:400, ab108394, Abcam, Cambridge, UK), ApoE (1:200, hybridoma supernatant, clone 26C11), IBA1 (1:500, ab5076, Abcam, Cambridge, UK), IBA1 (1:500, 234308, Synaptic Systems, Göttingen, Germany), NLRP3 (1:50, BSS-BS-10021R-100, Biozol Diagnostics Vertrieb, Eching, Germany) and CD68 (1:500, MCA1957GA, Bio-Rad, Feldkirchen, Germany).

Techniques: Generated, Transgenic Assay, Control, Staining, Two Tailed Test