Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

doi: 10.1161/ATVBAHA.120.314586

Figure Lengend Snippet: Figure 1. The SM22α-Cre specificity in the mouse brain vasculature. mT/mG reporter mice were bred with SM22α-Cre deleter mice (mT/mG:SM22α-Cre), and brain tissues were harvested at postnatal day 6 (P6). A, Sections of cerebellum and cerebrum were detected for mG expression by fluorescence microscopy. SM22α-Cre-driven mG was specifically detected in microvessels of both cerebrum and cerebellum (arrowheads) but not in control mT/mG mice (arrows). High power images of arrowhead-indicated regions are shown on the right. n=3 mice per group. B, Cerebral sections were immunostained with anti- PDGFR-β followed by an allophycocyanin (APC)-conjugated secondary antibody with an IgG isotype as a control. mG expression was colocalized with the pericyte (PC) marker PDGFR-β in the brain microvasculature of mT/mG:SM22α-Cre (arrowhead), but not in the IgG staining or in the control mT/mG mice (arrow). n=3 mice per group. C, mG+ and mG− cell populations were isolated from P6 mT/mG:SM22α- Cre brain tissues, and gene expression was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) with specific PC and endothelial cell (EC) markers as indicated. mG+ cells expressed PC marker genes PDGFRB (PDGFR-β, SCPG4 [NG-2], and ANPEP [CD13], but not EC marker genes PECAM1 [CD31], VEGFR2 [VEGFR2] and CDH5 [VE-cadherin]) with normalization by GAPDH. Data are mean±SEM; n=3; ***P<0.001 by unpaired 2-tailed Student t test. D and E, Mouse brain microvascular pericytes (mBMVPCs) and ECs (mBMVECs) were isolated from wild-type (WT) mice at P6, and immunostained with PC marker PDGFR-β and EC marker VE-cadherin. Phase images (D) and immunofluorescence images (E) are presented. F–I, Ccm3 deletion was specifically in mouse brain PCs but not in mouse brain ECs. mBMVPCs and mBMVECs were isolated from P6 WT and Ccm3smKO brain tissues. F, Cells were immunostained with PC marker PDGFR-β and EC marker VE-cadherin. G, Ccm3 gene expression was determined by qRT-PCR. n=3; ***P<0.001 by unpaired 2-tailed Student t test. H, CCM3 protein was determined by Western blotting. Representative blot form 3 experiments. I, CCM3 protein was determined by immunostaining using an anti-CCM3 antibody with costaining of antipaxillin antibody. n=3. Scale bar: 50 μm (A, B, and D); 25 μm (E and F); 10 μm (I).

Article Snippet: Human brain microvascular ECs (cAP0002) and human brain microvascular pericytes (hBMVPCs; cAP-0030) were purchased from Angio-Proteomie (Boston).

Techniques: Expressing, Fluorescence, Microscopy, Control, Marker, Staining, Isolation, Gene Expression, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Western Blot, Immunostaining

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

doi: 10.1161/ATVBAHA.120.314586

Figure Lengend Snippet: Figure 3 Continued. Representative images are shown in E. Quantification of % GFAP coverage on CD31+-vessel was quantified by Image J (F). G–I, Mouse brain microvascular pericytes (mBMVPCs) were isolated from neonatal WT and CCM3 smKO brains. 4×105 WT and CCM3- knockout (KO) mBMVPCs were seeded on fibronectin-coated culture slides for indicated times (0–16 h), and unattached cells were washed away. Cells were fixed with 4% paraformaldehyde (PFA) and stained with phalloidin (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue). Representative images for each time point are shown (G). Cell area (H) and cell length (I) were measured by Image J software. Ten fields were counted and n=3 repeated experiments. J and K, RNA-seq analyses. The endogenous CCM3 was knocked out by CRISPR/Cas9. mRNA from confluent WT and CCM3-KO human brain microvascular pericytes (hBMVPCs) were subjected to RNA-seq analyses. J, Gene expression value was estimated by Cufflinks (v1.2.0) and genes with >2-fold change between WT and KO were defined as differential expression. K, Gene Ontology analysis using GOstats was performed and the significant pathways (muscle cell migration and extracellular matrix [ECM] organization) are presented. n=2. Data are means±SEM. Scale bars: 25 μm (A, C, G, and I); 500 nm (E).

Article Snippet: Human brain microvascular ECs (cAP0002) and human brain microvascular pericytes (hBMVPCs; cAP-0030) were purchased from Angio-Proteomie (Boston).

Techniques: Isolation, Knock-Out, Staining, Software, RNA Sequencing, CRISPR, Gene Expression, Quantitative Proteomics, Migration

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

doi: 10.1161/ATVBAHA.120.314586

Figure Lengend Snippet: Figure 5. Cerebral cavernous malformation (CCM)3-knockout (KO) pericytes (PCs) attenuates PC migration and endothelial cell (EC)-PC interactions. CCM3-KO human brain microvascular pericytes (hBMVPCs) were re-expressed with vector (VC), CCM3-wild type (WT), or CCM3-4KE by lentivirus infection. A, WT, KO/VC, KO/CCM3-WT, and KO/CCM3-4KE hBMVPCs were harvested and subjected to Western blotting to test for adhesion complexes and RhoA-pMLC signaling. Relative protein levels were quantified and fold changes are presented by keeping WT as 1.0. B and C, 4×105 hBMVPCs were seeded on fibronectin-coated culture slides for 16 h. Cells were fixed with 4% paraformaldehyde (PFA) followed costaining with phosphor-paxillin (green) and phalloidin (red) with DAPI counterstaining (blue) (B). Number of FA per cell was measured by Image J software. Ten fields were counted and n=3 repeated experiments. D and E, Rescue PC migration by CCM3-WT but not by paxillin-defective CCM3-4KE mutant. WT, KO/VC, KO/CCM3-WT, and KO/CCM3-4KE hBMVPCs were subjected to wound injury followed by incubation for 24 h. D, Representative images of cell migration are shown. Dashed lines indicate the remaining gaps. E, Quantitation of EC migration. The percentage of unhealed wound was quantified, n=3. F and G, EC-PC interactions in 3-dimensional spheroid sprouting assay. Human brain microvascular ECs (hBMVECs) were infected with EGFP (enhanced green fluorescent protein)-expressing retroviruses, whereas WT, KO/VC, KO/CCM3-WT, and KO/CCM3-4KE hBMVPCs were infected with mCherry-expressing lentiviruses. ECs and PCs (2:1 ratio) were seeded to beads and coated with microbeads, embedded in fibrin gels and grown in EGM-2 endothelial growth medium for 4 d. A representative image of 10 beads for each sample is shown in F and percentage of PC coverage of sprouts is quantified in G. n=10, *P<0.05; **P<0.01 (1- way ANOVA). Additional 2 independent experiments were performed. Error bars indicate SEM. Scale bar: 10 μm (B); 100 μm (D and F).

Article Snippet: Human brain microvascular ECs (cAP0002) and human brain microvascular pericytes (hBMVPCs; cAP-0030) were purchased from Angio-Proteomie (Boston).

Techniques: Knock-Out, Migration, Plasmid Preparation, Infection, Western Blot, Software, Mutagenesis, Incubation, Quantitation Assay, Expressing

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

doi: 10.1161/ATVBAHA.120.314586

Figure Lengend Snippet: Figure 6. Cerebral cavernous malformation (CCM)3 loss in pericyte (PC) induces extracellular matrix (ECM) deposition in CCM. A and B, Increased ECM deposition in CCM3-deficient human brain microvascular pericytes (hBMVPCs). Wild-type (WT) and CCM3-KO hBMVPCs were cultured confluently on fibronectin-coated culture slides for 16 h. Cells were subjected to costaining with fibronectin (green) and Col IV (red) with DAPI counterstaining (blue). Mean fluorescence intensity (MFI)/per cell were measured by Image J software. Ten fields were counted and n=3 repeated experiments. C and D, P6 WT and Ccm3smKO mouse brain sections for staining of CD31 with fibronectin or NG-2. IgG isotype was used as a control. Representative images are shown (C). Normalized fibronectin MFI were measure by Image J (by taking WT as 1.0). Scale bar: 50 μm (A and C). E, A model for CCM3-depleted PC in promoting CCM lesion progression. The brain microvessels have an extraordinarily high PC to endothelial cell (EC) ratio, and PCs have multiple slender processes extending longitudinally to cover capillary EC for vascular integrity. Integrin-mediated matrix interactions and PC migration are critical in this process. Focal adhesion- mediated cell adhesion is important for cell migration, but the extent of adhesion can govern migration speed. We observed that CCM3- deficient PCs exhibit excess adhesion due to enhanced ECM deposition, ITG-β1 (integrin β1) activation and paxillin-mediated focal adhesion. We propose that CCM3 loss in PCs enhances PC adhesion while reducing PC protrusion and migration along EC, leading to the disruption of PC-EC interactions and resulting in CCM lesion formation.

Article Snippet: Human brain microvascular ECs (cAP0002) and human brain microvascular pericytes (hBMVPCs; cAP-0030) were purchased from Angio-Proteomie (Boston).

Techniques: Cell Culture, Fluorescence, Software, Staining, Control, Migration, Activation Assay, Disruption

Journal: Journal of vascular research

Article Title: Pericyte Dynamics during Angiogenesis: New Insights from New Identities

doi: 10.1159/000362276

Figure Lengend Snippet: Pericyte dynamics involved in angiogenesis. Pericyte dynamics include recruitment, ECM modulation, and growth factor presentation (secretion and binding). During recruitment, nearby pericytes or interstitial cell populations (i.e. fibroblasts or resident precursor cells – depicted as the blue cell) migrate to the endothelial cell-lined capillary sprout. Pericytes can also directly interact with endothelial cells via NG2 transmembrane protein binding to β1 integrins to influence their behavior [29, 36, 89]. Each dynamic represents a specific pericyte function critical for regulating capillary sprouting.

Article Snippet: Commercially available human brainderived vascular pericytes from ScienCell positively labeled for class III β-tubulin when cultured in Pericyte Medium containing 2% fetal bovine serum, 1% pericyte growth supplements and 1% 100x penicillin/streptomycin. ( A ; scale bar = 50 µm).

Techniques: Binding Assay, Protein Binding

Journal: Journal of vascular research

Article Title: Pericyte Dynamics during Angiogenesis: New Insights from New Identities

doi: 10.1159/000362276

Figure Lengend Snippet: Pericyte marker heterogeneity across a microvascular network. Examples of NG2 and SMA labeling along arterioles (A), venules (V) and capillaries (c) in adult rat mesenteric networks undergoing angiogenesis. NG2 labeling identifies SMA positive SMCs along arterioles, but not SMA positive SMCs along venules. At the capillary level, pericytes are positive for NG2 and negative for SMA. Scale bars = 25 µm.

Article Snippet: Commercially available human brainderived vascular pericytes from ScienCell positively labeled for class III β-tubulin when cultured in Pericyte Medium containing 2% fetal bovine serum, 1% pericyte growth supplements and 1% 100x penicillin/streptomycin. ( A ; scale bar = 50 µm).

Techniques: Marker, Labeling

Journal: Journal of vascular research

Article Title: Pericyte Dynamics during Angiogenesis: New Insights from New Identities

doi: 10.1159/000362276

Figure Lengend Snippet: Do pericytes change their phenotype during angiogenesis? Pericytes play a role in vessel stabilization during unstimulated (non-angiogenic) scenarios and capillary sprouting during pro-angiogenic scenarios. The question remains whether a subpopulation of pericytes emerge to perform functions unique to angiogenesis.

Article Snippet: Commercially available human brainderived vascular pericytes from ScienCell positively labeled for class III β-tubulin when cultured in Pericyte Medium containing 2% fetal bovine serum, 1% pericyte growth supplements and 1% 100x penicillin/streptomycin. ( A ; scale bar = 50 µm).

Techniques:

Journal: Journal of vascular research

Article Title: Pericyte Dynamics during Angiogenesis: New Insights from New Identities

doi: 10.1159/000362276

Figure Lengend Snippet: Pericyte differentiation during angiogenesis. This represents an example of the changing of phenotype by pericytes from an unstimulated (non-angiogenic) to an angiogenic scenario in adult rat mesenteric microvascular networks. In unstimulated microvascular networks, class III β-tubulin labeling is nerve specific and does not identify perivascular cells along PECAM positive arterioles, venules (V), or capillaries (c). During angiogenesis, perivascular cells along all vessels change their phenotype to become class III β-tubulin positive (arrows). The transient upregulation of class III β-tubulin by pericytes during angiogenesis highlights the potential for specialized pericyte subpopulations. Scale bars = 25 µm.

Article Snippet: Commercially available human brainderived vascular pericytes from ScienCell positively labeled for class III β-tubulin when cultured in Pericyte Medium containing 2% fetal bovine serum, 1% pericyte growth supplements and 1% 100x penicillin/streptomycin. ( A ; scale bar = 50 µm).

Techniques: Labeling

Journal: Journal of vascular research

Article Title: Pericyte Dynamics during Angiogenesis: New Insights from New Identities

doi: 10.1159/000362276

Figure Lengend Snippet: Pericytes in vitro express class III β-tubulin. Commercially available human brainderived vascular pericytes from ScienCell positively labeled for class III β-tubulin when cultured in Pericyte Medium containing 2% fetal bovine serum, 1% pericyte growth supplements and 1% 100x penicillin/streptomycin. (A; scale bar = 50 µm). B Higher magnification images display class III β-tubulin localization in tubule structures (Scale bar = 20 µm). C All human brainderived pericytes co-expressed the pericyte marker NG2 (Scale bar = 50 µm).

Article Snippet: Commercially available human brainderived vascular pericytes from ScienCell positively labeled for class III β-tubulin when cultured in Pericyte Medium containing 2% fetal bovine serum, 1% pericyte growth supplements and 1% 100x penicillin/streptomycin. ( A ; scale bar = 50 µm).

Techniques: In Vitro, Labeling, Cell Culture, Marker

Journal: Journal of vascular research

Article Title: Pericyte Dynamics during Angiogenesis: New Insights from New Identities

doi: 10.1159/000362276

Figure Lengend Snippet: Class III β-tubulin regulates pericyte migration. A–C Class III β-tubulin and DAPI nucleic acid labeling in the human brain-derived pericytes 6 hours after a scratch wound was applied (scale bars = 200 µm). Inhibition of class III β-tubulin was confirmed qualitatively by the reduction in class III β-tubulin positive cells (C) compared to control groups (A, B). D Quantification of scratch closure for the Control Sham, Control siRNA, and class III β-tubulin Target siRNA groups. At 72 hours (24 hour transfection + 48 hours for gene suppression to manifest), each monolayer was scratched and imaged. After 6 hours, scratches were again imaged and the change in scratch area was blindly quantified for 8 wells per group (n=8 per group).Scratch wound closure fractions were compared using a one-way ANOVA followed by a Student-Newman-Keuls pairwise comparison test. *, + represent significant differences (p<0.05) from Control Sham and Control siRNA groups, respectively. E Quantification of the fraction of class III β-tubulin positive cells inside the initial scratch area compared to the general population outside the scratch area (n= 8 per group). Statistical comparison between the two groups was made using a Student’s t-test. * represents a significant difference (p<0.05). F Example of class III β-tubulin and DAPI nucleic acid labeling along the leading of a scratch-wound in the Target siRNA group. Class III β-tubulin positive versus negative cells were more prone to migrate into the wound area. * indicates class III β-tubulin negative cell nuclei. Arrows indicate class III β-tubulin positive cells (scale bar = 50 µm). Values are presented as means +/− SEM.

Article Snippet: Commercially available human brainderived vascular pericytes from ScienCell positively labeled for class III β-tubulin when cultured in Pericyte Medium containing 2% fetal bovine serum, 1% pericyte growth supplements and 1% 100x penicillin/streptomycin. ( A ; scale bar = 50 µm).

Techniques: Migration, Labeling, Derivative Assay, Inhibition, Transfection

Journal: eLife

Article Title: Statistical analysis supports pervasive RNA subcellular localization and alternative 3' UTR regulation

doi: 10.7554/eLife.87517

Figure Lengend Snippet: ( a ) Experimental setup for human pericyte cell culture with reverse-transcriptase quantitative PCR (RT-qPCR) and extracellular TIMP3 protein ELISA readouts at four-timepoints. ( b ) TIMP3 protein secretion per cell per hour does not significantly change throughout culture time, even though the total protein measured by BCA does change. ( c ) qPCR experiment design with proximal and distal qPCR primers to distinguish long and short 3’ UTR isoforms. The proximal qPCR primer can detect both long and short isoforms while the distal primer can only amplify the long 3’ UTR. ( d ) The ratio of distal to proximal primer-template abundances significantly decreases throughout culture time, implying increased usage of the short TIMP3 3’ UTR compared to the long isoform. ( e ) TIMP3 3’ UTR abundance, normalized by 18 s housekeeper abundance, fluctuates from halving to doubling between culture timepoints for both distal and proximal primers.

Article Snippet: Human brain vascular pericytes (PCs, Sciencell) were cultured up to passage 5 in low-glucose DMEM (Gibco) supplemented with 10% FBS.

Techniques: Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Statistical analysis supports pervasive RNA subcellular localization and alternative 3' UTR regulation

doi: 10.7554/eLife.87517

Figure Lengend Snippet:

Article Snippet: Human brain vascular pericytes (PCs, Sciencell) were cultured up to passage 5 in low-glucose DMEM (Gibco) supplemented with 10% FBS.

Techniques: Software, Sequencing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity

doi: 10.1177/0271678X17720816

Figure Lengend Snippet: Pericytes share glucose with astrocytes in an occludin-modulated manner. (a) Confocal microscopy of a representative live pericyte-astrocyte co-culture 30 min after plating. Pericytes were pre-loaded with 2-NBDG (green; e.g. blue thick arrows) and astrocytes with violet-BMQC (cell mask, red) but not with 2-NBDG. Colocalization of both signals (yellow-orange; e.g. white thin arrows) indicated that astrocytes had received 2-NBDG. Intensity coefficient (Ic) represents the whole 2-NBDG fluorescence intensity normalized to the surface it occupies, regardless of cell type. A larger Ic means more 2-NBDG was introduced into the system (taken up by pericytes). Transfer coefficient (Tc) represents the fraction of astrocytic surface occupied by 2-NBDG normalized against the Intensity coefficient. A larger Tc implicates more 2-NBDG was distributed across all possible astrocytes, and represents greater transferred volumes. Data correspond to the quantitation of the images shown. They are representative of three separate experiments. (b) Distribution of 2-NBDG in the same co-culture shown in (a), 24 h after plating. Pericytes are devoid of any stain (e.g. white thin arrows). 2-NBDG signal (green) colocalizing with astrocytes is seen as cyan/white (e.g. yellow thick arrows). (c) Similar co-culture as in (a) recorded 30 min post-plating; however, pericytes were treated with negative-control siRNA (SCR) before being loaded with 2-NBDG. (d) Distribution of 2-NBDG (green/cyan) in the same co-culture shown in (c), 24 h post-plating. (e) Similar co-culture as in (a) and (c), recorded 30 min post-plating; however, pericytes were treated with anti-occludin siRNA (OCC−) before being loaded with 2-NBDG. (f) Distribution of 2-NBDG (green/cyan) in the same co-culture shown in (e), 24 h post-plating. All images are representative of three separate experiments. (g) Average intensity (Ic) and H) Transfer (Tc) coefficients depicting transcellular glucose transport between pericytes and astrocytes as shown in (a) to (c). n = 3, p vs. WT.

Article Snippet: Cell culture Primary human brain capillary pericytes and astrocytes (ScienCell, Carlsbad, CA, USA) were cultured in 5% CO 2 at 37°C in pericyte or astrocyte growth medium (ScienCell), following standard cell culture procedures, and used between passages 2 and 7.

Techniques: Confocal Microscopy, Co-Culture Assay, Fluorescence, Quantitation Assay, Staining, Negative Control

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity

doi: 10.1177/0271678X17720816

Figure Lengend Snippet: Pericytes share mitochondria with astrocytes in an occludin-mediated manner. (a) Live co-culture, 24 h post-plating, of astrocytes labeled with violet-BMQC (blue) and mitochondria-stained (TMRE in green) pericytes treated with anti-occludin siRNA (OCC−), negative-control siRNA (SCR), or non-treated (wild-type, WT). Thin white arrows exemplify TMRE-stained pericytes, while thick yellow arrows show pericyte mitochondria in the body of astrocytes (cyan signal). (b) Quantitation of TMRE intensities in astrocytes and pericytes in the same co-cultures shown in (a). Average ± SEM, n = 25 collected from three experiments, p vs. SCR. Only significant values are shown (c) Astrocytes treated with vehicle (Veh) or with endosulfan sulfate (ES, 4 h) to block their energetic metabolism. Middle image, surviving astrocytes exhibiting widened bodies and gross morphological alterations are exemplified by thin white arrows. Yellow arrowheads point to astrocytes that still retain their normal morphology. Right image: astrocytes treated with ES; however, isolated murine live brain capillaries (D shows a single brain capillary) were added to their growth medium 2 h post-treatment, and incubated for two additional hours. Note markedly improved astrocyte morphology. (e) Not-labeled human astrocytes cultured with murine live brain capillaries pre-labeled with TMRE (red) and 2-NBDG (green) for 2 h. TMRE and 2-NBDG transferred from microvessels to astrocytes (arrows) indicate transfer of mitochondria and glucose, respectively. (f) Similar TMRE and 2-NBDG transfer (arrows) in microvessel-rescued/ES-treated astrocytes after incubation with TMRE and 2-NBDG-labeled murine live brain capillaries.

Article Snippet: Cell culture Primary human brain capillary pericytes and astrocytes (ScienCell, Carlsbad, CA, USA) were cultured in 5% CO 2 at 37°C in pericyte or astrocyte growth medium (ScienCell), following standard cell culture procedures, and used between passages 2 and 7.

Techniques: Co-Culture Assay, Labeling, Staining, Negative Control, Quantitation Assay, Blocking Assay, Isolation, Incubation, Cell Culture

Journal: Experimental Hematology & Oncology

Article Title: ProNGF promotes brain metastasis through TrkA/EphA2 induced Src activation in triple negative breast cancer cells

doi: 10.1186/s40164-023-00463-6

Figure Lengend Snippet: proNGF enhances trans-BBB migration through Src activation independently of TrkA phosphorylation in TNBC. A Schematic representation of human BBB in vitro model, this one is characterized by a luminal compartment that allow culture of human BLEC on insert filtered (3 µm) and an abluminal compartment with plated human brain pericyte. Cancer cells are load with CellTracker™ before being incubated in the upper side. B-E Representative overlay ( B & D ) and associated quantification ( C & E ) of CellTracker™ stained MDA-MB-231-TrkA or CellTracker™ stained MDA-MB-231-TrkA-KD (green) on the lower compartment after a 16 h trans-BBB migration. ( F ) Emission ratio images of the ECFP/YPet-based Src biosensor during MDA-MB-231-TrkA-KD trans-BBB migration under abluminal proNGF stimulation. Data in B - E are representative of 3 independents experiments with all condition realized in duplicate. Data in C and E are represented by min to max interleaved box graph with median (black line). Two-way ANOVA followed by Tukey’s test for D and F . ***P ≤ 0.001, ****P ≤ 0.0001, ns (non-significant). For B and D scale bar = 100 µm, for D and F scale bare = 15 µm

Article Snippet: Human brain pericytes (Cosmo Bio Co., Ltd, Japan) are seeded in Petri dishes (100 mm diameter, Corning) coated with collagen I (100 μg/mL, Corning) and maintained in Dulbecco’s Modified Eagle Medium (DMEM, Life technologies) containing D-glucose (4.5 g/L) supplemented with 10% (v/v) FBS, penicillin (40 IU/mL), streptomycin (40 IU/mL) and L-glutamine (2 mM, Merck).

Techniques: Migration, Activation Assay, In Vitro, Incubation, Staining