human brain endothelial cell line  (Cedarlane)

Journal: Nature Communications

Article Title: Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2

doi: 10.1038/s41467-025-68058-9

Figure Lengend Snippet: A , Experimental timeline for inflammatory insult with TNF-α and FGF2 co-treatment. Once confluent (4 days), HBEC-5i or bEnd.3 were pretreated with FGF2 or vehicle for one hour and then stimulated with TNF-α or vehicle for up to 7 days. FGF2 alters mouse ( B ) and human ( C ) endothelial transcription in response to acute TNF-α stimulation. FGF2 promotes faster restoration of TNF-α-induced Cldn5 loss in mouse ( D ) and human ( E ) endothelial cells. Chronic stimulation with TNF-α leads to a reduction in endothelial monolayer integrity measured by trans-endothelial electrical resistance (TEER) in mouse ( F ) and human ( G ) endothelial cells. FGF2 co-treatment preserves normal TEER despite TNF-α. H 7 days of TNF-α treatment promotes spikes and discontinuities in Cldn5 tight junction strands in bEnd.3, which is reversed by FGF2 treatment (scalebar = 20 μm). Data represent mean ± s.e.m., and each experiment was replicated at least twice on independent samples. Group comparisons were evaluated with two-way ANOVA followed by Bonferroni’s post hoc tests; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The human brain microvascular endothelial cell line HBEC-5i (ATCC CRL-3245, male donor according to https://www.cellosaurus.org/CVCL_4D10 ) and the mouse brain endothelial cell line bEnd.3 (ATCC CRL-2299) were subcultured and stored in banks at −150 ° C. Cells were thawed as needed and cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 25 ug/mL gentamicin (Gibco), and 1X endothelial cell growth supplement (ScienCell).

Techniques:

Journal: Nature Communications

Article Title: Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2

doi: 10.1038/s41467-025-68058-9

Figure Lengend Snippet: A 1 h pretreatment with Fgf2 increases serine-9 phosphorylation of GSK3β in HBEC-5i when compared to no treatment (CTRL 0 h). TNF-α treatment induces rapid, transient dephosphorylation of GSK3β, but this effect is not reversed by Fgf2 coadministration. Each dot represents a replicate ( n = 3). B 1 h Fgf2 pretreatment diminishes basal β-catenin phosphorylation when compared to no treatment (CTRL 0 h). Further, while TNF-α induces a rapid reduction in phosphorylated β-catenin, Fgf2 reverses this dynamic upon inflammatory activation ( n = 3). C In health control endothelial cells (top), β-catenin interacts with VE-Cadherin at the cell membrane, and this complex inhibits Cldn5 transcriptional suppression by FOXO1. Excess cytosolic β-catenin is phosphorylated by GSK3β, targeting it for degradation. When stimulated with TNFα, unbound β-catenin complexes with FOXO1, leading to suppression of Cldn5 expression (bottom, red arrow), while a small amount is targeted for degradation. Meanwhile, when FGF2 is co-administered with TNF-α (bottom, blue arrow), our results suggest that unbound β-catenin is strongly redirected toward GSK3β-mediated phosphorylation. D 30 min of TNF-α is sufficient to induce β-catenin distribution at tight junctions ( n = 4 replicates) with representative images on the right ( E ) (scalebar = 20 μm). F Fgf2 attenuates TNF-α-induced reductions in the wound healing capacity of HBEC-5i ( n = 4 replicates) (**** p < 0.0001). Data represent mean ± s.e.m., and each experiment was replicated at least twice on independent samples. Group comparisons were evaluated with one or two-way ANOVA followed by Bonferroni’s post hoc tests or two-tailed t-tests with Welch’s correction when appropriate; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The human brain microvascular endothelial cell line HBEC-5i (ATCC CRL-3245, male donor according to https://www.cellosaurus.org/CVCL_4D10 ) and the mouse brain endothelial cell line bEnd.3 (ATCC CRL-2299) were subcultured and stored in banks at −150 ° C. Cells were thawed as needed and cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 25 ug/mL gentamicin (Gibco), and 1X endothelial cell growth supplement (ScienCell).

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Activation Assay, Control, Membrane, Expressing, Two Tailed Test

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/D3 cells treated with Stx1a or Stx2a exhibited prominent morphological alterations when observed under a fluorescence microscope at 20X magnification, whereas cells treated with Stx1a mut or Stx2a mut did not show such changes. Images were collected from cells incubated with or without Stxs for 24, 48, and 72 h; ( B ) hCMEC/D3 cells were seeded in 6-well plates (5.0 ×10 5 cells/well) and incubated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) or Stx2a mut (10 ng/ml) for 24, 48, 72 h. Cell viability was determined using WST-8 assays, expressed as percentage viability and fold change relative to untreated controls ( left panel ). hCMEC/D3 cells (1.0 × 10 5 cells/well) were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) and Stx2a mut (10 ng/ml) for 24 h, and caspase-3/7 activity was measured using the caspase Glo-3/7 assay. Under the same conditions, hCMEC/D3 cells (5.0 × 10 5 cells/well) were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) and Stx2a mut (10 ng/ml) for 24 h, and then protein samples were subjected to Western blotting using an anti-cleaved caspase-3 antibody ( right panel ). β-Actin was used as a control for equal protein loading. The results are a representative experiment obtained from three independent experiments; ( C ) Gb3 expression in hCMEC/D3 cells was detected by staining with Alexa Fluor 647-conjugated anti-CD77/Gb3 antibody or isotype control (mouse IgM-Alexa Fluor 647) for 1 h at 4oC. Representative results from three independent experiments are shown. Statistical significance. Asterisks indicate significant differences between control cell values and Stxs-treated cells (Panel B). * p < 0.05, ** p < 0.01, *** p < 0.001 versus control.

Article Snippet: The human brain endothelial cell line hCMEC/D3 (Merck) was cultured in Endothelial Cell Growth Medium (PromoCell, Germany) supplemented with 1% Antibiotic-Antimycotic (Thermo Fisher Scientific, USA), and human astrocyte cell line A172 was cultured in RPMI 1640 medium (Corning, Thermo Fisher Scientific) supplemented with 10% FBS and 1% Antibiotic-Antimycotic (Thermo Fisher Scientific) at 37°C in a humidified incubator containing 5% CO 2 . hCMEC/D3 and A172 cells were seeded at 5.0 × 10 5 cells/well into 6-well plates, washed once with sterile Dulbecco's phosphate-buffered saline (DPBS) (Sigma-Aldrich, USA), and treated with 10 ng/ml Stx2a in Endothelial Cell Growth Medium or RPMI containing 0.5% FBS without supplements for the indicated time periods.

Techniques: Fluorescence, Microscopy, Incubation, Activity Assay, Caspase-Glo Assay, Western Blot, Control, Expressing, Staining

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/D3 cells were seeded in 6-well plates (5.0 × 10 5 cells/well). After washing with culture medium, cells were fixed and nuclei were stained with DAPI reagent. Representative DAPI-positive cells were visualized by fluorescence microscopy. To detect Stx translocation to the ER, cells were stimulated with complete growth medium containing 50 nM ER-tracker (red) live cell staining dye 2 h after treatment with Alexa Fluor 488-conjugated Stx1a (100 ng/ml). After washing, cells were captured using a fluorescence microscope EVOS M5000. Yellow fluorescence indicates co-localization of Stx1a with the ER marker. The scale bars represent 150 μm. The bar graph represents the mean ± SEM of the Pearson's correlation coefficient for the co-localization of Stx1-Alexa Fluor 488/ER tracker. At least 30 cells per condition were analyzed. Asterisks indicate statistically significant differences between the control and Stx1-treated groups. *** = p < 0.001; ( B ) hCMEC/D3 cells were stimulated with Stx2a (10 ng/ml) for 0, 3, 6, 9, 12 and 24 h. After washing, cells were lysed at the indicated time points, and the presence of activated ER stress sensors and downstream targets in the cell lysates was determined by Western blotting. Untreated cells served as controls, and β-actin was used as a loading control; ( C ) hCMEC/D3 cells were treated as described above, and CHOP and DR5, spliced XBP1/unspliced XBP1 mRNA expression was measured by RT-qPCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH); ( D ) hCMEC/D3 cells were stimulated with Stx2a (10 ng/ml) for 0, 24, 48, and 72 h. At the indicated time points, cells were lysed, and the expression of ROS-related markers (Nrf-2, HO-1, SOD-2, NO2) in cell lysates was measured by RT-qPCR and normalized using GAPDH. Asterisks indicate significant differences between control cell values and Stxs-treated cells (Panels C and D) at each time point. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Data are shown as mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Article Snippet: The human brain endothelial cell line hCMEC/D3 (Merck) was cultured in Endothelial Cell Growth Medium (PromoCell, Germany) supplemented with 1% Antibiotic-Antimycotic (Thermo Fisher Scientific, USA), and human astrocyte cell line A172 was cultured in RPMI 1640 medium (Corning, Thermo Fisher Scientific) supplemented with 10% FBS and 1% Antibiotic-Antimycotic (Thermo Fisher Scientific) at 37°C in a humidified incubator containing 5% CO 2 . hCMEC/D3 and A172 cells were seeded at 5.0 × 10 5 cells/well into 6-well plates, washed once with sterile Dulbecco's phosphate-buffered saline (DPBS) (Sigma-Aldrich, USA), and treated with 10 ng/ml Stx2a in Endothelial Cell Growth Medium or RPMI containing 0.5% FBS without supplements for the indicated time periods.

Techniques: Staining, Fluorescence, Microscopy, Translocation Assay, Marker, Control, Western Blot, Expressing, Quantitative RT-PCR

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/D cells were seeded in 6-well plates at a density of 5.0 × 10 5 cells/well and treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx1B (100 ng/ml), Stx2a (10 ng/ml), Stx2a mut (10 ng/ml), or Stx2B (10 ng/ml) for 3 h. After 3 h, cells were washed and cell lysates were collected. Phosphorylation of p38, JNK, and ERK were assessed by Western blotting. β-Actin was used as a loading control. The graphs show the mean ± SEM of band densities normalized by the division of β-Actin band densities and compared to untreated control cell values ( right panel ). Statistical analyses of densitometric scans from at least three independent experiments are shown; ( B ) hCMEC/D3 cells were seeded in 6-well plates at a total cell density of approximately 5.0 × 10 5 cells/well and treated with Stxs for 0, 24, 48, and 72 h. At each time point, cells were lysed and mRNA expression levels of inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α, CXCL1, and CCL2) were measured by RT-qPCR and normalized to GAPDH; ( C ) Under the same conditions, supernatants were collected and protein levels of IL-1β, IL-6, IL-8, TNF-α and CCL2 were quantified by ELISA using kit standards. Data are presented as mean ± SEM from three independent experiments. Asterisks indicate significant differences between control and Stx-treated cells (Panels B, C) at the indicated time points. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The human brain endothelial cell line hCMEC/D3 (Merck) was cultured in Endothelial Cell Growth Medium (PromoCell, Germany) supplemented with 1% Antibiotic-Antimycotic (Thermo Fisher Scientific, USA), and human astrocyte cell line A172 was cultured in RPMI 1640 medium (Corning, Thermo Fisher Scientific) supplemented with 10% FBS and 1% Antibiotic-Antimycotic (Thermo Fisher Scientific) at 37°C in a humidified incubator containing 5% CO 2 . hCMEC/D3 and A172 cells were seeded at 5.0 × 10 5 cells/well into 6-well plates, washed once with sterile Dulbecco's phosphate-buffered saline (DPBS) (Sigma-Aldrich, USA), and treated with 10 ng/ml Stx2a in Endothelial Cell Growth Medium or RPMI containing 0.5% FBS without supplements for the indicated time periods.

Techniques: Phospho-proteomics, Western Blot, Control, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/D3 cells were seeded in 6-well plates at a total cell density of approximately 5.0 × 10 5 cells/well and treated with Stxs for 0, 3, 6, 9, 12, and 24 h. At each time point, cells were lysed, and mRNA expression of ZO-1, CLDN3, Occludin and JAM2 was measured by RT-qPCR. Expression levels were normalized to GAPDH; ( B ) hCMEC/D3 cells were seeded as above and treated with Stx2a for 0, 6, 12, 24, and 48 h. At each time point, cells were lysed, and the tight junction proteins CLDN1, ZO-1, and Ecadherin were analyzed by Western blotting. β-Actin was used as a loading control; ( C ) hCMEC/D3 cells were seeded in the insert wells of Trans-well plates at 3.0 × 10 5 cells/well. Cells were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml), or Stx2a mut (10 ng/ml) for 24 h. Fluorescein-conjugated 45kDa ovalbumin was added to the toxin-treated insert wells, and supernatants were collected from the lower chamber after 1 h. Cell permeability was evaluated by measuring the fluorescence of ovalbumin translocated from the insert wells to the lower chamber using a microplate reader. Asterisks indicate significant differences between the control cell values and the Stxs-treated cells (Panels A, C) at each time point. * = p < 0.05; ** = p < 0.01; ***= p < 0.001.

Article Snippet: The human brain endothelial cell line hCMEC/D3 (Merck) was cultured in Endothelial Cell Growth Medium (PromoCell, Germany) supplemented with 1% Antibiotic-Antimycotic (Thermo Fisher Scientific, USA), and human astrocyte cell line A172 was cultured in RPMI 1640 medium (Corning, Thermo Fisher Scientific) supplemented with 10% FBS and 1% Antibiotic-Antimycotic (Thermo Fisher Scientific) at 37°C in a humidified incubator containing 5% CO 2 . hCMEC/D3 and A172 cells were seeded at 5.0 × 10 5 cells/well into 6-well plates, washed once with sterile Dulbecco's phosphate-buffered saline (DPBS) (Sigma-Aldrich, USA), and treated with 10 ng/ml Stx2a in Endothelial Cell Growth Medium or RPMI containing 0.5% FBS without supplements for the indicated time periods.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Permeability, Fluorescence

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/ D cells were seeded in 6-well plates. Cells were pretreated with z-VAD (20 μM) for 1 h before Stx2a exposure and divided into the following groups: untreated control, Stx2a-treated, z-VAD only, and Stx2a + z-VAD pre-treated. After 24 h of Stx2a treatment, cell viability was assessed using the WST-8 assay ( left panel ). Subsequently, Western blot analysis showed that the total levels of caspase-3/7 in the cell lysates were decreased only in the Stx2a-treated group. Protein samples were prepared using anti-caspase-3, anti-cleaved caspase-3, and anti-β-Actin antibodies ( right panel ). β-Actin was used as a control for equal protein loading; ( B ) hCMEC/D3 cells were seeded as described above and treated with z-VAD 1 h before Stx2a treatment. Cell lysates were harvested from the following groups: untreated control, Stx2a-treated, z-VAD only, and Stx2a + z-VAD pre-treated. Culture supernatants were collected, and the levels of secreted IL-1β, IL-6, IL-8, TNF-α and CCL2 were measured using ELISA ( left panel ). Total RNA was isolated from cell lysates, and mRNA expression levels of IL-1β, IL-6, IL-8, TNF-α, CXCL1, and CCL2 were quantified using RT-qPCR ( right panel ). RNA expression levels were normalized using GAPDH; ( C ) hCMEC/D3 cells were seeded in 6-well plates at a total cell density of 5.0 × 10 5 cells/well and treated with NAC (5μM) 1 h before Stx2a exposure. The experimental groups included control, Stx2a-treated, NAC only, and Stx2a + NAC pre-treated. After 24 h of Stx2a treatment, the cytotoxicity level was measured by LDH assay using the cell culture supernatant. mRNA expression levels of Nrf-2, HO-1, and SOD-2 were measured by RT-qPCR and normalized to GAPDH, and the degree of NO expression was measured by NO assay; ( D ) hCMEC/D3 cells were seeded in 6-well plates at a density of 5.0 × 10 5 cells/well and treated with z-VAD and NAC 1 h before Stx2a treatment. Cell lysates were harvested from the following groups: control, Stx2a-treated, z-VAD + Stx2a, and NAC + Stx2a groups. After 24 h of Stx2 treatment, washed cell lysates were harvested, and the mRNA expression of ZO-1, CLDN3, Occludin and JAM2 were measured using RT-qPCR. RNA expression levels were normalized using GAPDH. Asterisks indicate significant differences compared to the control group, Stx-treated group, or between monotherapy vs. combination therapy (e.g., z-VAD alone vs. z-VAD+Stx2a) (Panels A, B) and compared to the control group, Stx-treated group, or between monotherapy vs. pre-treatment therapy ( e.g. , NAC alone vs. NAC+Stx2a) (Panel C). Significant differences are indicated between the control and cells treated with Stx, Stx+z-VAD, or Stx+NAC (Panel D).* = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Article Snippet: The human brain endothelial cell line hCMEC/D3 (Merck) was cultured in Endothelial Cell Growth Medium (PromoCell, Germany) supplemented with 1% Antibiotic-Antimycotic (Thermo Fisher Scientific, USA), and human astrocyte cell line A172 was cultured in RPMI 1640 medium (Corning, Thermo Fisher Scientific) supplemented with 10% FBS and 1% Antibiotic-Antimycotic (Thermo Fisher Scientific) at 37°C in a humidified incubator containing 5% CO 2 . hCMEC/D3 and A172 cells were seeded at 5.0 × 10 5 cells/well into 6-well plates, washed once with sterile Dulbecco's phosphate-buffered saline (DPBS) (Sigma-Aldrich, USA), and treated with 10 ng/ml Stx2a in Endothelial Cell Growth Medium or RPMI containing 0.5% FBS without supplements for the indicated time periods.

Techniques: Control, Western Blot, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Quantitative RT-PCR, RNA Expression, Lactate Dehydrogenase Assay, Cell Culture

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/D3 cells were seeded in the insert wells of Trans-well plates at 3.0 × 10 5 cells/well, and A172 cells were seeded in the bottom wells of Trans-well plates at 5.0 × 10 5 cells/well. The insert wells containing hCMEC/D3 cells were stimulated with Stx2a (10 ng/ml) for 24 h. After 24 h, the supernatants were collected and cytotoxicity in A172 cells was assessed by LDH assay ( left panel ). A representative flow-cytometry readout of Gb3 in A172 astrocytes ( right panel ). ( B ) Subsequently, the A172 cells in the bottom wells were washed, and the cell lysates were harvested and the protein levels of caspase-3 and cleaved caspase-3 were determined by Western blotting. Anti-caspase-3, anticleaved caspase-3, and anti-β-Actin antibodies were used for protein samples. β-Actin served as a loading control ( C-D ) Culture supernatants of A172 cells seeded in the same manner as above were harvested, and the secretion levels of IL-1β, IL-6, IL-8, TNF-α, and CCL2 were determined using ELISA. Subsequently, the mRNA expression levels of IL-1β, IL-6, IL-8, TNF-α, CXCL1 and CCL2 were assessed by using RT-qPCR in cell lysates. RNA expression levels were normalized using GAPDH. Asterisks indicate significant differences between the control group and cells treated with Stx, Stx+z-VAD, and Stx+NAC (Panels A, C, D). * = p < 0.05; ** = p < 0.01; ***= p < 0.001.

Article Snippet: The human brain endothelial cell line hCMEC/D3 (Merck) was cultured in Endothelial Cell Growth Medium (PromoCell, Germany) supplemented with 1% Antibiotic-Antimycotic (Thermo Fisher Scientific, USA), and human astrocyte cell line A172 was cultured in RPMI 1640 medium (Corning, Thermo Fisher Scientific) supplemented with 10% FBS and 1% Antibiotic-Antimycotic (Thermo Fisher Scientific) at 37°C in a humidified incubator containing 5% CO 2 . hCMEC/D3 and A172 cells were seeded at 5.0 × 10 5 cells/well into 6-well plates, washed once with sterile Dulbecco's phosphate-buffered saline (DPBS) (Sigma-Aldrich, USA), and treated with 10 ng/ml Stx2a in Endothelial Cell Growth Medium or RPMI containing 0.5% FBS without supplements for the indicated time periods.

Techniques: Lactate Dehydrogenase Assay, Flow Cytometry, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, RNA Expression

Journal: Journal of Neuroinflammation

Article Title: Transcriptomic profiling of thrombus-derived extracellular vesicles reveals PECAM-1 as a potential inflammatory marker for cardioembolic stroke patients

doi: 10.1186/s12974-025-03555-8

Figure Lengend Snippet: Effect of oxLDL and thrombo-inflammatory stimuli on the mRNA expression of selected candidates in human brain microvascular endothelial cells (hCMEC/D3). Expression of PECAM-1 (panel A ), IL-8 (panel B ), ITGB1 (panel C ), CD44 (panel D ), and SLC3A2 (panel E) in hCMEC/D3 cell cultures stimulated with oxLDL for 24 h, or with TNFα, IL1β and thrombin for 6 h and 12 h. Data are expressed as fold-change (FC) relative to control. * p < 0.05, ** p < 0.01, *** p < 0.001 vs baseline

Article Snippet: Human brain microvascular endothelial cell line (hCMEC/D3, SC0066, Merck), was seeded on 0.7 mg/mL rat collagen type I in PBS (Sigma-Aldrich, 08–115) coated 75 cm 2 flask and cultured in complete EndoGROTM-MV medium (Merck, SCME004) supplemented with 1% penicillin and streptomycin (PS, 100 U/mL and 100 μg/mL, Sigma-Aldrich).

Techniques: Expressing, Control