Journal: BMC Neuroscience

Article Title: CSF from Parkinson disease Patients Differentially Affects Cultured Microglia and Astrocytes

doi: 10.1186/1471-2202-11-151

Figure Lengend Snippet: Photographs of astrocyte cultures in which diverse morphology changes were not evident between the PD-CSF, control and diseases control CSF groups . With the addition of disease (neurological) control CSF, a higher growth rate was seen than that in PD-CSF treated cells, but a lower growth rate than in the control (no-CSF) treated cultures. Cell count per frame is in lower left corner; TNTC = too numerous to count. Magnification ×100.

Article Snippet: Approximately 2.0 × 10 6 Human Astrocytes (Cat# HAST 040, Clonexpress, Gaithersburg, MD) and Human Glialblastoma cells, U-118 MG (Cat# HTB-15, ATCC, Manassas, VA) were treated with 300 μl of 1× sample buffer (2X = 10.0 ml of 10% SDS, 4.0 ml 1 M Tris-Cl pH = 6.8, 7.5 ml Glycerol and 45.5 ml ddH2O) diluted 1:1 in 1× PBS and boiled for 5 minutes.

Techniques: Control, Cell Counting

Journal: BMC Neuroscience

Article Title: CSF from Parkinson disease Patients Differentially Affects Cultured Microglia and Astrocytes

doi: 10.1186/1471-2202-11-151

Figure Lengend Snippet: α-Synuclein density in astrocytes, α-Synuclein distribution patterns were not different between treatment groups . Red = α-synuclein; blue = DAPI (nuclei); green = filamentous actin. Magnification ×600.

Article Snippet: Approximately 2.0 × 10 6 Human Astrocytes (Cat# HAST 040, Clonexpress, Gaithersburg, MD) and Human Glialblastoma cells, U-118 MG (Cat# HTB-15, ATCC, Manassas, VA) were treated with 300 μl of 1× sample buffer (2X = 10.0 ml of 10% SDS, 4.0 ml 1 M Tris-Cl pH = 6.8, 7.5 ml Glycerol and 45.5 ml ddH2O) diluted 1:1 in 1× PBS and boiled for 5 minutes.

Techniques:

Journal: BMC Neuroscience

Article Title: CSF from Parkinson disease Patients Differentially Affects Cultured Microglia and Astrocytes

doi: 10.1186/1471-2202-11-151

Figure Lengend Snippet: Histogram demonstrating changes in α-synuclein content of cultured astrocytes of all treatment groups over the course of the experiment . On day 7 there was a significant (p < 0.05) increase in α-synuclein content in the PD-CSF treated astrocytes, compared to the neurological disease control CSF treated cells, but all cells showed a trend to a return to baseline of α-synuclein content (n = 8). Western blot of protein levels is included for comparison.

Article Snippet: Approximately 2.0 × 10 6 Human Astrocytes (Cat# HAST 040, Clonexpress, Gaithersburg, MD) and Human Glialblastoma cells, U-118 MG (Cat# HTB-15, ATCC, Manassas, VA) were treated with 300 μl of 1× sample buffer (2X = 10.0 ml of 10% SDS, 4.0 ml 1 M Tris-Cl pH = 6.8, 7.5 ml Glycerol and 45.5 ml ddH2O) diluted 1:1 in 1× PBS and boiled for 5 minutes.

Techniques: Cell Culture, Control, Western Blot, Comparison

Journal: BMC Neuroscience

Article Title: CSF from Parkinson disease Patients Differentially Affects Cultured Microglia and Astrocytes

doi: 10.1186/1471-2202-11-151

Figure Lengend Snippet: Microglia models showing α-synuclein locations . 3D models of stacked deconvoluted acquisitions to show the initial peripheral localization of α-synuclein in microglia (Image A), which becomes aggregated and peri-nuclear or juxta-nuclear following PD-CSF treatment (Image B). Astrocytes (Image C), still demonstrate peripheral α-synuclein and minimal aggregation, even after seven days of treatment (Magnification × 900).

Article Snippet: Approximately 2.0 × 10 6 Human Astrocytes (Cat# HAST 040, Clonexpress, Gaithersburg, MD) and Human Glialblastoma cells, U-118 MG (Cat# HTB-15, ATCC, Manassas, VA) were treated with 300 μl of 1× sample buffer (2X = 10.0 ml of 10% SDS, 4.0 ml 1 M Tris-Cl pH = 6.8, 7.5 ml Glycerol and 45.5 ml ddH2O) diluted 1:1 in 1× PBS and boiled for 5 minutes.

Techniques:

Journal: PLoS ONE

Article Title: Ibudilast, a Pharmacologic Phosphodiesterase Inhibitor, Prevents Human Immunodeficiency Virus-1 Tat-Mediated Activation of Microglial Cells

doi: 10.1371/journal.pone.0018633

Figure Lengend Snippet: A , Human microglial cells (1×10 5 ) were left untreated (NT) or were treated with Tat (100 nM) for 8 h with or without pre-treatment for 30 min. with increasing concentrations of ibudilast or vehicle (Veh), as indicated. TNFα, IL-1β, IL-6, and MCP-1 levels in culture supernatants were analyzed by Multi-Plex cytokine array, as described in . The Tat+Veh-treated samples were set to 100% and all other samples were compared to this value. Results are shown as mean ± SD of values derived from two replicates from a single representative experiment. Statistical significance (***, p<0.001; *, p<0.05) is indicated, as compared to Tat+Veh-treated cells. The average cytokine/chemokine concentration of the Tat+Veh-treated sample was as follows; TNFα: 8166 pg/mL, IL-1β: 3.3 pg/mL, IL-6: 6027 pg/mL, and MCP-1: 1480 pg/mL. B , Similarly, murine microglial cells (BV-2; 1.2×10 5 ) were left untreated (NT) or were treated with Tat (100 nM) for 8 h with or without pre-treatment for 30 min. with increasing concentrations of ibudilast or vehicle (Veh), as indicated. TNFα release was measured by ELISA. Results are shown as mean ± SEM of values derived from three replicates from a single representative experiment; three total experiments were performed. Statistical significance (p<0.001) is indicated, as compared to Tat+Veh-treated cells (***). The TNFα concentration of the Tat+Veh-treated sample was 1861 pg/mL. C , BV-2 cells (1.2×10 5 ) were left untreated (NT) or were treated with increasing concentrations of ibudilast or vehicle (Veh), as indicated, for 8 h. The MTT assay was used as a measure of cell viability. Percent survival was calculated as compared to the untreated sample. Results are shown as mean ± SEM of values derived from four replicates from a single representative experiment; two total experiments were performed.

Article Snippet: Human microglial cells that were isolated from fetal human brain were obtained from Clonexpress (Gaithersburg, MD, USA) and were maintained in 50∶50 DMEM: F-12 supplemented with 5% FBS and 10 ng/mL of M-CSF.

Techniques: Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, MTT Assay