pericyte basal growth medium  (PromoCell)

Journal: Histochemistry and Cell Biology

Article Title: EBF1 is expressed in pericytes and contributes to pericyte cell commitment

doi: 10.1007/s00418-021-02015-7

Figure Lengend Snippet: EBF1 plays a key role in the pericyte phenotype cell commitment. a Significant downregulation of EBF1 mRNA levels (about 75% decrease; p < 0.01) was obtained in HBVPs by means of siRNA technology, measured by RT-qPCR 24 h after transfection (upper histogram). Immunoblotting performed with protein extracts confirmed the reduction in the synthesis of EBF1 protein (about 50% decrease; p < 0.05). The representative image on the right shows the corresponding EBF1 band at 66 kDa. b Histogram shows that after EBF1 silencing, the expression of EBF3, a marker linked to the MSC phenotype, is significantly increased ( p < 0.05). c Silencing of EBF1 does not affect cell proliferation as assessed by the evaluation of the nuclear antigen Ki-67 expression by RT-qPCR, 24 h after seeding cells with or without the addition of different proliferative stimuli, such as the medium from U87 glioblastoma cells cultured under hypoxic conditions or the complete culture medium containing PGS and 10% FBS. d We then investigated whether EBF1 silencing affects the phenotype of the pericytes. Indeed, the histograms show a significant reduction in the main pericyte markers PDGFRβ ( p < 0.05) at both the transcriptional ( p < 0.05) and protein ( p < 0.01) levels. e Additionally, we found a significant reduction in the pericyte marker CD146 ( p < 0.01). The expression levels of CD90, a marker linked to a mesenchymal phenotype, did not show a significant reduction. f Of note, the expression levels of VEGF, Ang-1, NG2 and TGF-β, cytokines produced by pericytes during different phases of angiogenesis are significantly reduced in EBF1-silenced HBVPs ( p < 0.05). Overall, data confirmed a functional role of EBF1 in pericyte cell fate commitment and functionality. * p < 0.05, ** p < 0.01. CTRL (control); siRNA (EBF1-silenced); SCR (scramble, control siRNA); Basal (basal conditions); Hypoxic (hypoxic conditions)

Article Snippet: After transfection, the medium was replaced with the following media: basal pericyte medium (Sciencell) without supplementation; pericyte medium with the addition of Pericyte Growth Supplement (PGS) and 2% fetal bovine serum (FBS); pericyte medium with 50 μL/mL of medium obtained from U87 glioblastoma cells cultured for 48 h under hypoxic conditions.

Techniques: Quantitative RT-PCR, Transfection, Western Blot, Expressing, Marker, Cell Culture, Produced, Functional Assay, Control

Journal: Stem cells (Dayton, Ohio)

Article Title: A-Kinase Anchor Protein 12 is required for oligodendrocyte differentiation in adult white matter

doi: 10.1002/stem.2771

Figure Lengend Snippet: (a) Immunostaining showed that AKAP12 was not expressed in OPCs (PDGF-R-α positive cells) in the corpus callosum region in wild-type mouse brain sections. Instead, AKAP12 was confirmed to be expressed in pericytes (PDGF-R-β positive cells) in the corpus callosum region in wild-type mouse brain sections. Importantly, AKAP12-expressing pericytes were closely located to OPCs in the corpus callosum region. Bar=100 μm. (b) Double-staining of PDGF-R-β and AKAP12 using confocal microscopy confirmed that pericytes express AKAP12. Bar=20 μm. Please see Suppl Figure S6 for additional data from immunostaining with another pericyte marker CD13.

Article Snippet: Human brain vascular pericytes [ 30 ] were cultured in pericyte basal medium (Sciencell research laboratories) containing 2% fetal bovine serum and pericyte growth supplement (Sciencell research laboratories) onto poly-l-lysine-coated plates.

Techniques: Immunostaining, Expressing, Double Staining, Confocal Microscopy, Marker

Journal: Stem cells (Dayton, Ohio)

Article Title: A-Kinase Anchor Protein 12 is required for oligodendrocyte differentiation in adult white matter

doi: 10.1002/stem.2771

Figure Lengend Snippet: (a) Cultured pericytes expressed AKAP12 and the AKAP12 expression was suppressed by AKAP12 siRNA. β-actin was used as an internal control. (b) AKAP12 knockdown did not affect pericyte survival assessed by LDH assay. LDH amounts in the culture media were not different between control-siRNA-treated pericytes and AKAP12-siRNA-treated pericytes. Values are mean ± SD. N=5.

Article Snippet: Human brain vascular pericytes [ 30 ] were cultured in pericyte basal medium (Sciencell research laboratories) containing 2% fetal bovine serum and pericyte growth supplement (Sciencell research laboratories) onto poly-l-lysine-coated plates.

Techniques: Cell Culture, Expressing, Control, Knockdown, Lactate Dehydrogenase Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: A-Kinase Anchor Protein 12 is required for oligodendrocyte differentiation in adult white matter

doi: 10.1002/stem.2771

Figure Lengend Snippet: Proteome Profiler™ Human XL Cytokine Array using pericyte-conditioned-media showed that AKAP12 knockdown changed the pattern of secreting factors from pericytes. LIF, BDNF, GRO-α, and HGF are known to promote OPC differentiation. Those factors were downregulated in the AKAP12-deficient pericyte cultures. Please see Suppl Figure S8 for other factors from pericyte cultures.

Article Snippet: Human brain vascular pericytes [ 30 ] were cultured in pericyte basal medium (Sciencell research laboratories) containing 2% fetal bovine serum and pericyte growth supplement (Sciencell research laboratories) onto poly-l-lysine-coated plates.

Techniques: Knockdown

Journal: Stem cells (Dayton, Ohio)

Article Title: A-Kinase Anchor Protein 12 is required for oligodendrocyte differentiation in adult white matter

doi: 10.1002/stem.2771

Figure Lengend Snippet: Cultured OPCs were maintained in control media, pericyte-conditioned media (P-CM), or conditioned media from AKAP12-deficient pericytes (P−CMAKAP12-). Five days later, those OPCs were used for immunostaining or western blotting analyses. (a-b) Immunostaining with anti-MBP (oligodendrocyte marker) antibody showed that P-CM increased the number of MBP−positive cells, but P-CMAKAP12- showed less effect on OPC maturation. Bar=100 μm. Values are mean ± SD. N=5. *P<0.05 vs control, and #P<0.05 vs P-CM. (c-d) The levels of MBP expression in P-CM-treated OPCs were also lower compared to ones in P−CMAKAP12- -treated OPCs. Values are mean ± SD. N=4. *P<0.05 vs P-CM.

Article Snippet: Human brain vascular pericytes [ 30 ] were cultured in pericyte basal medium (Sciencell research laboratories) containing 2% fetal bovine serum and pericyte growth supplement (Sciencell research laboratories) onto poly-l-lysine-coated plates.

Techniques: Cell Culture, Control, Immunostaining, Western Blot, Marker, Expressing