Journal: ASN Neuro

Article Title: Gas6 Enhances Axonal Ensheathment by MBP+ Membranous Processes in Human DRG/OL Promyelinating Co-Cultures

doi: 10.1042/an20130022

Figure Lengend Snippet: Figure 1 Generation of human fetal DRG explants (A and B) with parallel arrays of NF + axons, and enriched OPCs from mixed glial cultures (C and D) (A) Axons radiating from a DRG explant maintained in culture for 3 weeks. (B) NF + -stained axons radiating from a DRG explant. (C and D) Enriched OPCs from human fetal brain maintained in the presence (C) and absence (D) of Gas6 are βIV tubulin + (green) and express Olig1 (red) in the nucleus following 20 days in culture. Blue is Hoechst stain. An Olympus digital microscope was used.

Article Snippet: Recombinant human Gas6 (Amgen) grown in vitamin Kcontaining medium was measured using the human Gas6 Quantikine ELISA kit (DGAS60, R&D Systems) following the manufacturer’s protocol.

Techniques: Staining, Microscopy

Journal: ASN Neuro

Article Title: Gas6 Enhances Axonal Ensheathment by MBP+ Membranous Processes in Human DRG/OL Promyelinating Co-Cultures

doi: 10.1042/an20130022

Figure Lengend Snippet: Figure 2 Human OPC/DRG co-cultures in medium containing PDGF + Gas6 express MBP + processes that align and elongate along NF + axons, while OLs in co-cultures maintained in BDNF + IGF1 + Gas6 have more mature MBP + processes that ensheath axons Immunofluorescent staining and confocal microscopy show MBP + OLs (green) and NF + axons (red; 1:2000; chick IgY-cy3) captured on an Olympus Digital microscope after 14 days in co-culture; scale bar for all images is 50 μm. (A) Co-cultures maintained in PDGF medium minus Gas6. (B) Co-cultures main- tained in PDGF medium plus Gas6 have MBP + OL processes aligned with axons. (C and E) Co-cultures maintained in IGF1 + BDNF-defined medium minus Gas6. (D and F) Co-cultures maintained in IGF1 + BDNF-defined medium plus Gas6. Scale bar in (A) designates magnification for all panels.

Article Snippet: Recombinant human Gas6 (Amgen) grown in vitamin Kcontaining medium was measured using the human Gas6 Quantikine ELISA kit (DGAS60, R&D Systems) following the manufacturer’s protocol.

Techniques: Staining, Confocal Microscopy, Microscopy, Co-Culture Assay

Journal: ASN Neuro

Article Title: Gas6 Enhances Axonal Ensheathment by MBP+ Membranous Processes in Human DRG/OL Promyelinating Co-Cultures

doi: 10.1042/an20130022

Figure Lengend Snippet: Figure 3 Gas6 enhances OL wrapping and mean process length in human co-cultures For experiments 1 and 2, co-cultures were maintained in defined medium as defined in the DRG/co-culture section of the Materials and methods (DMEM supplemented with 2% B27, 1% N2, 20 ng/ml rhBDNF, 20 ng/ml IGF1, 40 ng/ml T3, 40 ng/ml T4, 5ng/ml NT3, antibiotic/antimycotic, plus and minus Gas6). (A) Experiment 1, multiple, randomly selected MBP + fields were photographed. From each random field, the percentage of MBP + oligodendrocytes with processes wrapping axons relative to the total number of MBP + oligodendrocytes/field was calculated. (B) Experiment 2, MBP + OLs with obvious processes aligned with an axon were photographed, and the percentage of MBP + OLs with processes wrapping axons relative to the total number of MBP + OLs/field was calculated. (C) Histogram depicts the mean length (μm) of MBP + processes wrapping NF + axons in Gas6 −and Gas6 + co-cultures; P = 0.007; Mann–Whitney test.

Article Snippet: Recombinant human Gas6 (Amgen) grown in vitamin Kcontaining medium was measured using the human Gas6 Quantikine ELISA kit (DGAS60, R&D Systems) following the manufacturer’s protocol.

Techniques: Co-Culture Assay, MANN-WHITNEY

Journal: ASN Neuro

Article Title: Gas6 Enhances Axonal Ensheathment by MBP+ Membranous Processes in Human DRG/OL Promyelinating Co-Cultures

doi: 10.1042/an20130022

Figure Lengend Snippet: Figure 4 Multiple MAG + MBP + OL processes from a single OL can ensheath the same axon and multiple axons Co-cultures were maintained in defined medium plus and minus Gas6 for 14 days (see the Materials and methods section). (A–L) Immunofluorescent staining and light microscopy. MAG + (red), MBP + (green) OLs, NF + (blue) axons; scale bar = 10 μm. (M and N) Z-series and confocal microscopy shows wrapping of MAG- and MBP-positive processes. Maximal projection of MBP + processes was obtained in Volocity. Linear line of a box corresponds to 1 unit = 24 μm. Backgrounds were adjusted to the negative primary antibody isotype controls. For example, the MAG primary antibody (IgG1) was substituted with primary antibody MOPC-31C purified clone for mouse IgG1 (BD Pharmingen; 557273).

Article Snippet: Recombinant human Gas6 (Amgen) grown in vitamin Kcontaining medium was measured using the human Gas6 Quantikine ELISA kit (DGAS60, R&D Systems) following the manufacturer’s protocol.

Techniques: Staining, Light Microscopy, Confocal Microscopy

Journal: ASN Neuro

Article Title: Gas6 Enhances Axonal Ensheathment by MBP+ Membranous Processes in Human DRG/OL Promyelinating Co-Cultures

doi: 10.1042/an20130022

Figure Lengend Snippet: Figure 5 MBP + OL processes (green) spiral and ensheath multiple NF + axons (red) in co-cultures maintained for 14 days in defined medium minus (a and b) and plus (c) Gas6 Identification of multiple Z-series images captured by confocal microscopy (Leica) and analyzed in Volocity. The arrow in the image a shows an MBP + OL process spirally wrapping the axon. Images in (a and b) show MBP + OLs wrapping NF + axons in co-cultures maintained in defined medium only. (c) A representative image from a Gas6-treated co-culture. Arrowheads in (b and c) indicate areas where MBP + (green) membranes totally ensheath the axon (red).

Article Snippet: Recombinant human Gas6 (Amgen) grown in vitamin Kcontaining medium was measured using the human Gas6 Quantikine ELISA kit (DGAS60, R&D Systems) following the manufacturer’s protocol.

Techniques: Confocal Microscopy, Co-Culture Assay

Journal: ASN Neuro

Article Title: Gas6 Enhances Axonal Ensheathment by MBP+ Membranous Processes in Human DRG/OL Promyelinating Co-Cultures

doi: 10.1042/an20130022

Figure Lengend Snippet: Figure 6 Multiple OL/axonal contacts are observed in Gas6-treated co-cultures A co-culture maintained for 14 days in defined medium plus Gas6 was embedded in Epon, and uranyl-stained and 1-μm sections were examined by EM. Examination of grids found fields containing axons with multiple oligodendrocytes. Scale bars in (A–D) are 1 μm; the scale bar in (E) is 0.5 μm.

Article Snippet: Recombinant human Gas6 (Amgen) grown in vitamin Kcontaining medium was measured using the human Gas6 Quantikine ELISA kit (DGAS60, R&D Systems) following the manufacturer’s protocol.

Techniques: Co-Culture Assay, Staining

Journal: ASN Neuro

Article Title: Gas6 Enhances Axonal Ensheathment by MBP+ Membranous Processes in Human DRG/OL Promyelinating Co-Cultures

doi: 10.1042/an20130022

Figure Lengend Snippet: Figure 7 Visualization of wrapping of axons in human co-cultures in the presence of Gas6 (2.6 nM) (A) Two axons (AX) wrapped in myelin membrane. (B) Upper axon from (A) shows wrapping of one myelin membrane encircling the other (*); higher magnification * is shown in (E). (C) Lower axon from (A) shows OL membrane wrapping and multiple contacts/adhesions between the axonal membrane and the OL membrane; higher magnifications of the contacts are shown in (F) and (G). (D) OL membrane wrapping an axon, and contacting an additional membrane; higher magnification is shown in (H). Scale bars are 1 μm.

Article Snippet: Recombinant human Gas6 (Amgen) grown in vitamin Kcontaining medium was measured using the human Gas6 Quantikine ELISA kit (DGAS60, R&D Systems) following the manufacturer’s protocol.

Techniques: Membrane