PMC

Cultures of hOPCs were treated during 24(A) Microphotographs showing Olig2 and Sox2 positive cells in control and 0G treated cultures. Scale bar = 50 µm. (B) The percentage of positive cells in each experimental condition was analyzed by confocal microscopy. Results are the means ± SEM for three independent experiments. *p<0.05, **p<0.01, versus respective control.

hOPCs were incubated in a chamber with 5% CO2 at 37°C, which was placed on the stage of a spinning disc confocal inverted microscope. Cells were imaged at 6 min intervals for a period of 26 h. Estimated cell cycle times and percentage of mitotic hOPCs for control and 0G treated cultures are shown. Values are expressed as mean ± SEM of four independent experiments. *p<0.05, **p<0.01, versus respective control.

Cultures of hOPCs were treated during 24 and 48-four hour pulses of 10 µM bromo-deoxyuridine (BrdU) were done during the 0G treatment as well as in parallel untreated cultures. After each BrdU pulse, cells were fixed and immunostained with anti-BrdU. (A) Microphotographs showing BrdU positive cells at 24 and 48 h. green: BrdU immunostaining. Scale bar = 50 µm. (B) The percentage of BrdU positive cells in each experimental condition was compared with respective controls. Values are expressed as mean ± SEM of two independent experiments. ***p<0.001 versus control.

Mixed glial cultures of PLP-GFP labeled OPCs were treated during 24, 48 and 72 h in 0G. (A) Yellow arrowheads define PLP-GFP/Sox2 double positive cells at 24, 48 and 72 h. Green: PLP-GFP, Red: Sox2 immunostaining. Scale bar = 50 µm. (B and C) After treatment, cells were fixed and immunostained for several oligodendrocyte stage markers and the percentage of positive cells in each experimental condition was analyzed by confocal microscopy. (D) Mixed glial cultures of PLP-GFP labeled OPCs were treated during 10, 15 and 30 days in 0G. After treatment, cells were fixed and immunostained for Olig1 and Olig2 and the percentage of positive cells in each experimental condition was analyzed by confocal microscopy. Results are the means ± SEM for three independent experiments. *p<0,05, **p<0,01 and ***p<0,001 vs. respective controls.

Mixed glial cultures of PLP-GFP labeled OPCs were treated during 24, 48 and 72 h in 0G. Twenty-four hour pulses of 10 µM bromo-deoxyuridine (BrdU) were begun at 0 h, 24 h and 48 h. After each BrdU pulse, cells were fixed and immunostained with anti-BrdU and anti-NG2 antibodies. (A) Microphotographs showing NG2+/BrdU+ cells at 24, 48 and 72 h. Green: NG2 immunostaining, and Red: BrdU immunostaining. Scale bar = 50 µm. (B and C) The percentage of NG2+/BrdU+ and PLP-GFP+/BrdU+ cells in each experimental condition was compared with respective controls. Values are expressed as mean ± SEM of two independent experiments. *p<0.05, **p<0.01 versus respective control.

(A) Mixed glial cultures of PLP-GFP labeled OPCs were incubated in a chamber with 5% CO2 at 37°C, which was placed on the stage of a spinning disc confocal microscope. Pictures show examples of living GFP-labeled OPCs that were imaged for a period of 26 h. Scale bar = 50 µm. In the insert, a single migrating OPC is shown, small yellow arrowheads point at the leading process and the cell soma is shown by the large arrowhead. Scale bar = 50 µm (B) The average leading process length and soma area of migrating OPCs was calculated from at least 50 cells in each experimental condition. Values are expressed as mean ± SEM of at least four independent experiments. *p<0.05, **p<0.01 versus control cells.

hOPCs were imaged at 6(A) Time-lapse series of hOPCs from cultures treated during 24 h in 0G. Yellow arrowheads identify a migrating hOPC. Each frame represents a single section of a time lapse video sequence. Time is denoted in hours in the bottom right corner. Scale bar = 50 µm. Cell migration speed and distances were analyzed off-line by tracing individual cells at different times, after which migratory values were statistically analyzed across the experimental conditions. (B) hOPC migration speed was calculated from at least 60 cells in each experimental condition. (C and D) Total migration distance was followed for 12 h in 50 cells from each experimental condition and the percentage of migrating cells was calculated from the entire cell population. Values are expressed as mean ± SEM of at least four independent experiments. **p<0.01, ***p<0.001 versus control cells.

Mixed glial cultures of PLP-GFP labeled OPCs were incubated in a chamber with 5% CO2 at 37°C, which was placed on the stage of a spinning disc confocal microscope. GFP-labeled OPC clones were imaged with a specific GFP filter at 6 min intervals for a period of 26 h. (A) Time-lapse series of OPC∼GFP clones from cultures treated during 24 h in 0G. Yellow arrowheads designate cytokinesis events. Tracking of cells between birth cytokinesis and division cytokinesis was noted with a yellow asterisk near the cell, which was generated from frame to frame. Each frame represents a single section of a time lapse video sequence. Time is denoted in hours in the bottom right corner. Scale bar = 50 µm. (B and C) Estimated cell cycle times and percentage of mitotic OPCs for each experimental condition. Values are expressed as mean ± SEM of four independent experiments. *p<0.05, **p<0.01, versus respective control.

Mixed glial cultures of PLP-GFP labeled OPCs were imaged with a GFP filter at 6 min intervals for a period of 16 h. (A) Time-lapse series of OPCs from cultures treated during 24 h in 0G. Yellow arrowheads identify a migrating OPC. Each frame represents a single section of a time lapse video sequence. Time is denoted in hours in the bottom right corner. Scale bar = 50 µm. Cell migration speed and distances were analyzed off-line by tracing individual cells at different times, after which migratory values were statistically analyzed across the experimental conditions. (B) OPC migration speed was calculated from at least 60 cells in each experimental condition. (C and D) Total migration distance was followed for 12 h in 50 cells from each experimental condition and the percentage of migrating cells was calculated from the entire cell population. Values are expressed as mean ± SEM of at least four independent experiments. **p<0.01, ***p<0.001 versus control cells.