Journal: BMC Neuroscience

Article Title: Modelling physiological and pathological conditions to study pericyte biology in brain function and dysfunction

doi: 10.1186/s12868-018-0405-4

Figure Lengend Snippet: Culture media alters pericyte phenotype and proliferation. Human brain pericytes were plated at a low density and allowed to proliferate in either DMEM/F12 with 10% FBS or Pericyte Medium. Brightfield imaging of pericyte morphology was performed 3 and 7 days post plating ( a ). Pericytes were then fixed and the nuclei stained with Hoechst. The average nuclei area ( b ) and the total cell count ( c ) were determined by automated image analysis. Five days after plating, 10 µM EdU was added to select wells for an additional 48 h. Cells were fixed and nuclei counterstained with Hoechst ( d ). The percentage of EdU positive cells was determined by automated image analysis ( e ). Data are displayed as mean ± SEM of three independent experiments. ***p < 0.001 versus DMEM/F12 cultured pericytes (Student’s t test). Scale bar = 50 µm

Article Snippet: Here we sought to investigate how two commonly used culture media, high serum containing DMEM/F12 and low serum containing Pericyte Medium (ScienCell), altered the phenotype of human brain pericytes and neuroinflammatory responses.

Techniques: Imaging, Staining, Cell Counting, Cell Culture

Journal: BMC Neuroscience

Article Title: Modelling physiological and pathological conditions to study pericyte biology in brain function and dysfunction

doi: 10.1186/s12868-018-0405-4

Figure Lengend Snippet: Culture media modifies expression of typical pericyte markers. Human brain pericytes were plated at a low density and allowed to proliferate in either DMEM/F12 with 10% FBS or Pericyte Medium for 7 days. At completion, pericytes were fixed and immunostained for the pericyte-associated markers CD146, αSMA, P4H, desmin, fibronectin, NG2, PDGFRβ and COL-IV. Nuclei were counterstained with Hoechst ( a ). The integrated intensity/cell of pericyte marker expression was determined by automated image analysis ( b ). RNA was extracted from samples treated as above and qRT-PCR performed to determine pericyte marker gene expression ( c ). Data are displayed as mean ± SEM of three independent experiments. **p < 0.01; ***p < 0.001 versus DMEM/F12 cultured pericytes (Student’s t test). Scale bar = 50 µm

Article Snippet: Here we sought to investigate how two commonly used culture media, high serum containing DMEM/F12 and low serum containing Pericyte Medium (ScienCell), altered the phenotype of human brain pericytes and neuroinflammatory responses.

Techniques: Expressing, Marker, Quantitative RT-PCR, Gene Expression, Cell Culture

Journal: BMC Neuroscience

Article Title: Modelling physiological and pathological conditions to study pericyte biology in brain function and dysfunction

doi: 10.1186/s12868-018-0405-4

Figure Lengend Snippet: Culture media modifies IL-1β-induced transcription factor expression in human brain pericytes. Human brain pericytes were plated at a low density and allowed to proliferate in either DMEM/F12 with 10% FBS or Pericyte Medium for 7 days. For the final 1 h ( a ) or 24 h ( b ) 10 ng/mL IL-1β or vehicle was added. Pericytes were fixed and immunostained for the transcription factors NF-kB p65 ( a ) or C/EBPδ ( b ). Nuclei were counterstained with Hoechst. The percentage of cells displaying nuclear NF-kB p65 ( c ) or scored positive for C/EBPδ ( d ) was determined by automated image analysis. RNA was extracted from pericytes treated with 10 ng/mL IL-1β or vehicle (0.0001% BSA in PBS) for the final 24 h of a 7 day culture and the gene expression of CEBPD determined by qRT-PCR ( e ). Data are displayed as mean ± SEM of three independent experiments. NS = p > 0.05; **p < 0.01; ***p < 0.001 as designated or compared to vehicle control in respective media (Two-way ANOVA). Scale bar = 50 µm

Article Snippet: Here we sought to investigate how two commonly used culture media, high serum containing DMEM/F12 and low serum containing Pericyte Medium (ScienCell), altered the phenotype of human brain pericytes and neuroinflammatory responses.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Control

Journal: BMC Neuroscience

Article Title: Modelling physiological and pathological conditions to study pericyte biology in brain function and dysfunction

doi: 10.1186/s12868-018-0405-4

Figure Lengend Snippet: Culture media modifies IL-1β induced inflammatory mediator expression in human brain pericytes. Human brain pericytes were plated at a low density and allowed to proliferate in either DMEM/F12 with 10% FBS or Pericyte Medium for 7 days. For the final 24 h 10 ng/mL IL-1β or vehicle (0.0001% BSA in PBS) was added. Pericytes were fixed and immunostained for the inflammatory mediators ICAM-1 or MCP-1 ( a ). Nuclei were counterstained with Hoechst. The integrated intensity of ICAM-1/cell was determined by automated image analysis ( b ), the secretion of soluble ICAM-1 (sICAM-1) was determined by a cytometric bead array ( c ) and the gene expression of ICAM1 was determined by qRT-PCR ( d ). Similarly, the integrated intensity of MCP-1/cell was determined by automated image analysis ( e ), the secretion of MCP-1 was determined by a cytometric bead array ( f ) and the gene expression of MCP1 determined by qRT-PCR ( g ). Data are displayed as mean ± SEM from three independent experiments except CBA data which depicts mean ± SEM of triplicate wells from one representative case of three independent experiments. NS = p > 0.05; **p < 0.01; ***p < 0.001 as designated, or compared to vehicle control in respective media (Two-way ANOVA). Scale bar = 50 µm

Article Snippet: Here we sought to investigate how two commonly used culture media, high serum containing DMEM/F12 and low serum containing Pericyte Medium (ScienCell), altered the phenotype of human brain pericytes and neuroinflammatory responses.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Control

Journal: BMC Neuroscience

Article Title: Modelling physiological and pathological conditions to study pericyte biology in brain function and dysfunction

doi: 10.1186/s12868-018-0405-4

Figure Lengend Snippet: Culture media modifies phagocytic capacity of human brain pericytes. Human brain pericytes were plated at a low density and allowed to proliferate in either DMEM/F12 with 10% FBS or Pericyte Medium for 7 days. Cells were incubated with Fluoresbrite ® YG carboxylate microspheres of 1–2 µm diameter for 2 h and the phagocytic capacity of pericytes determined by flow cytometry. One representative plot for 1 µm beads ( a ) and 2 µm beads ( b ) is shown. The mean fluorescent intensity (MFI) from three independent experiments was determined ( c ). Pericytes were treated as above, except beads were incubated with cells for 24 h, fixed and nuclei counterstained with DRAQ5. Fluorescent microscopy images of one representative case from three independent experiments are shown ( d ). Data are displayed as mean ± SEM of three independent experiments. NS = p > 0.05; **p < 0.01; ***p < 0.001 (Student’s t test). Scale bar = 50 µm

Article Snippet: Here we sought to investigate how two commonly used culture media, high serum containing DMEM/F12 and low serum containing Pericyte Medium (ScienCell), altered the phenotype of human brain pericytes and neuroinflammatory responses.

Techniques: Incubation, Flow Cytometry, Microscopy

Journal: BMC Neuroscience

Article Title: Modelling physiological and pathological conditions to study pericyte biology in brain function and dysfunction

doi: 10.1186/s12868-018-0405-4

Figure Lengend Snippet: Culture media modifies pericyte migration following a scratch wound injury. Human brain pericytes were plated at a low density and allowed to proliferate in either DMEM/F12 with 10% FBS or Pericyte Medium for 7–14 days. The resulting confluent pericyte monolayer was scratched down the centre of the well using a sterile 200 µL pipette tip and wells were washed twice with PBS to remove detached cells. An equal number of wells were left unscratched. Pericytes were incubated for a further 48 h to allow migration into the scratch wound to occur. Cells were fixed and stained with Coomassie blue and imaged by bright field microscopy. Consistency of scratches between wells can be observed ( a ). A magnified image of a representative well shows the difference in pericyte migration ( b ). The gap area, defined as the percentage of each site devoid of Coomassie staining, was determined by automated image analysis ( c ). Data are displayed as mean ± SEM of three independent experiments. *p < 0.05; ***p < 0.001 (Two-way ANOVA with Bonferroni post-test). Scale bar = 50 µm

Article Snippet: Here we sought to investigate how two commonly used culture media, high serum containing DMEM/F12 and low serum containing Pericyte Medium (ScienCell), altered the phenotype of human brain pericytes and neuroinflammatory responses.

Techniques: Migration, Sterility, Transferring, Incubation, Staining, Microscopy

Journal: BMC Neuroscience

Article Title: Modelling physiological and pathological conditions to study pericyte biology in brain function and dysfunction

doi: 10.1186/s12868-018-0405-4

Figure Lengend Snippet: Culture medium does not alter pericyte responsiveness to growth factors TGFβ 1 and PDGF-BB. Human brain pericytes were plated at a low density and allowed to proliferate in either DMEM/F12 with 10% FBS or Pericyte Medium for 7 days. For the final 2 h pericytes were incubated with 10 ng/mL TGFβ 1 or vehicle (1 µM citric acid, pH 3 with 0.0001% BSA). Cells were fixed and immunostained for SMAD2/3 ( a ) and the nuclear intensity was determined by automated imagining analysis ( b ). Human brain pericytes were plated at a low density and allowed to proliferate in either DMEM/F12 with 10% FBS or Pericyte Medium for 7 days. For the final 30 min pericytes were treated with 10–100 ng/mL PDGF-BB or vehicle (4 µM hydrochloric acid with 0.0001% BSA). Cells were brought into suspension with Accutase and incubated with a PDGFRβ antibody or an isotype control. Cell surface PDGFRβ was determined by flow cytometry ( c ). Data are displayed as mean ± SEM of three independent experiments. ***p < 0.001 versus vehicle control in respective media (Two-way ANOVA). Scale bar = 50 µm

Article Snippet: Here we sought to investigate how two commonly used culture media, high serum containing DMEM/F12 and low serum containing Pericyte Medium (ScienCell), altered the phenotype of human brain pericytes and neuroinflammatory responses.

Techniques: Incubation, Suspension, Control, Flow Cytometry