cell culture human brain microvascular endothelial cells  (ATCC)

Journal: Nitric oxide : biology and chemistry

Article Title: Regulation of endothelial barrier integrity by redox-dependent nitric oxide signaling: Implication in traumatic and inflammatory brain injuries

doi: 10.1016/j.niox.2018.12.007

Figure Lengend Snippet: Human brain microvessel endothelial cells (hBMVECs) were treated with thrombin (0.1unit/ml) and time dependent activation of RhoA activity was analyzed (left panel). The cells were also treated with various concentrations of thrombin and a dose dependent activation of RhoA activation was analyzed at 5 min following the treatment as described in method section (A). hBMVECs were treated with various concentrations of thrombin and intracellular Ca2+ ([Ca2+]i) influx was analyzed by fluorometric assay as described in method section (B-i). Twenty five seconds following thrombin treatment, the increased [Ca2+]i influxes were represented by bar graph (B-ii). In another set of experiment, thrombin-induced time- and concentration-dependent phosphorylation of myosin light chain (Ser19) was analyzed in hBMVECs by Western analysis. β-actin was used for internal loading control for Western analysis (C). hBMVECs were treated with thrombin (0.1unit/ml for 30 min) and development of F-actin stress fiber was analyzed by immunofluorescent staining of F-actin bundles by Phalloidin (red) and phosphorylated MLC (p-MLC; green). Nuclei were stained by DAPI (blue) (D-i). For endothelial barrier study, hBMVECs cultured on transwell plates were analyzed for transendothelial electric resistance (TEER) in the absence or presence of thrombin (0.1unit/ml for 30 min) treatment (D-ii). To investigate causal relationships between RhoA activation or [Ca2+]i influx and MLC phosphorylation, hBMVECs were pretreated with RhoA inhibitor I (C3 transferase/C3-Tr; 1µg/ml) or [Ca2+]i chelator BAPTA (100µM) for 30min, followed by thrombin treatment (0.1unit/ml) for 10 min, and then cellular levels of phospho- and total-MLC levels were analyzed by Western analysis (E). The vertical bars (B-ii) and dots (D-ii) are means of individual data set (n=3) and T-bars are standard deviation. *** p ≤ 0.001 as compared to control group. All experiments were repeated at least three times and representative data are shown.

Article Snippet: Cell culture Primary human brain microvascular endothelial cells (hBMVECs) were purchased from Angio-Proteomie (Cat#: cAP-0002, Atlanta, GA).

Techniques: Activation Assay, Activity Assay, Concentration Assay, Phospho-proteomics, Western Blot, Control, Staining, Cell Culture, Standard Deviation

Journal: Nitric oxide : biology and chemistry

Article Title: Regulation of endothelial barrier integrity by redox-dependent nitric oxide signaling: Implication in traumatic and inflammatory brain injuries

doi: 10.1016/j.niox.2018.12.007

Figure Lengend Snippet: Cell lysates from cultured human brain microvessel endothelial cells (hBMVECs), neurons, and activated microglia were analyzed for expression levels of eNOS, nNOS, and iNOS (A-i). hBMVECs were treated with thrombin (0.1 unit/ml) and the cellular levels of NO was analyzed by fluorometric analysis using dye DAF-FM (A-ii). hBMVECs were treated with thrombin (0.1 unit/ml) and time course activation of eNOS was analyzed by Western analysis using antibody specific to phospho (Ser1177) eNOS (A-iii). β-actin was used for internal loading control and lysate extracted from glutamate treated cultured neurons was used for positive control for nNOS activation. hBMVEC were treated with thrombin and time and concentration dependent accumulation of protein-associated S-nitrosothiols (Pr-SNO) (B) or protein-associated 3-nitrotyrosine (N-Tyr) (C) or were analyzed by biotin switch assay or ELISA, respectively. The vertical columns represent means of individual data set and T-bars are standard deviation. ** p ≤ 0.01 and *** p ≤ 0.001 as compared to the control group. All experiments were repeated at least three times and representative data are shown.

Article Snippet: Cell culture Primary human brain microvascular endothelial cells (hBMVECs) were purchased from Angio-Proteomie (Cat#: cAP-0002, Atlanta, GA).

Techniques: Cell Culture, Expressing, Activation Assay, Western Blot, Control, Positive Control, Concentration Assay, Biotin Switch Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Nitric oxide : biology and chemistry

Article Title: Regulation of endothelial barrier integrity by redox-dependent nitric oxide signaling: Implication in traumatic and inflammatory brain injuries

doi: 10.1016/j.niox.2018.12.007

Figure Lengend Snippet: Human brain microvessel endothelial cells (hBMVECs) in the presence or absence of NOS inhibitor L-NIO (10µM; pretreated for 30min) were treated with thrombin (0.1 unit/ml for 5min) and MLC phosphorylation (Ser19) was analyzed by Western analysis with β-actin as internal loading control (A). hBMVECs were treated with thrombin (0.1 unit/ml for 20min) in the presence or absence of L-NIO (10µM; pretreated for 30min) or ONOO− scavenger FeTTPS (10µM; pretreated for 30min) and cellular levels of protein-associated 3-nitrotyrosine (a protein adduct formed by ONOO−) was analyzed by ELISA (B). hBMVECs were treated with thrombin (0.1 unit/ml for 5min) in the presence or absence of FeTPPS or L-NIO and MLC phosphorylation (C), RhoA activity (D), and intracellular Ca2+ ([Ca2+]i) influx (E) were analyzed. To investigate causal relationship between RhoA activation or [Ca2+]i influx and eNOS phosphorylation (Ser1177), hBMVECs were pretreated with RhoA inhibitor I (C3 transferase/C3-Tr; 1µg/ml) or [Ca2+]i chelator BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; 100µM] for 30min, followed by thrombin treatment (0.1unit/ml) for 10 min, then cellular levels of phospho and total eNOS levels were analyzed by Western analysis (F). To confirm the role of eNOS in regulation of MLC phosphorylation, hBMVECs were treated with NOS inhibitor L-NIO (10µM), in the presence or absence of DETA-NO (free NO donor; 1mM), for 30min and effect of thrombin (0.1 unit/ml for 5min) on MLC phosphorylation was analyzed by Western analysis. The vertical bars are means of individual data and T-bars are standard deviation. *** p ≤ 0.001 as compared to the control group. +++ p ≤ 0.001 as compared to thrombin treated group. All experiments were repeated at least three times and representative data are shown.

Article Snippet: Cell culture Primary human brain microvascular endothelial cells (hBMVECs) were purchased from Angio-Proteomie (Cat#: cAP-0002, Atlanta, GA).

Techniques: Phospho-proteomics, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Activity Assay, Activation Assay, Standard Deviation

Journal: Nitric oxide : biology and chemistry

Article Title: Regulation of endothelial barrier integrity by redox-dependent nitric oxide signaling: Implication in traumatic and inflammatory brain injuries

doi: 10.1016/j.niox.2018.12.007

Figure Lengend Snippet: Human brain microvessel endothelial cells (hBMVECs) were treated with various concentrations of GSNO or SIN-1 (ONOO− donor), incubated for 2hr, and cellular levels of S-nitrosylated proteins and RhoA (A-i) and tyrosine-nitrated proteins and RhoA (A-ii) were analyzed as described in method section. hBMVECs were treated with thrombin (0.1 unit/ml for 5min), in the presence or absence of various concentrations GSNO or SIN-1 (pretreated for 2hr), and RhoA activity was analyzed as described in method section (B). hBMVECs were treated with thrombin (0.1 unit/ml) in the presence or absence of various concentrations GSNO or SIN-1 and intracellular Ca2+ ([Ca2+]i) influx (C-i and ii) and cell viability (MTT assay) (C-iii) were analyzed. hBMVECs were treated with thrombin (0.1 unit/ml for 5min), in the presence or absence of various concentrations GSNO or SIN-1 (D-i) or decomposed GSNO (100µM) or SIN-1 (1000µM) (D-ii), and MLC phosphorylation was analyzed by Western analysis. β-actin was used for internal loading control for Western analysis. The vertical bars are means of individual data and T-bars are standard deviation. *** p ≤ 0.001 as compared to the control group. + p ≤ 0.05 and ++ p ≤ 0.01 as compared to thrombin treated group. All experiments were repeated at least three times.

Article Snippet: Cell culture Primary human brain microvascular endothelial cells (hBMVECs) were purchased from Angio-Proteomie (Cat#: cAP-0002, Atlanta, GA).

Techniques: Incubation, Activity Assay, MTT Assay, Phospho-proteomics, Western Blot, Control, Standard Deviation

Journal: Nitric oxide : biology and chemistry

Article Title: Regulation of endothelial barrier integrity by redox-dependent nitric oxide signaling: Implication in traumatic and inflammatory brain injuries

doi: 10.1016/j.niox.2018.12.007

Figure Lengend Snippet: A. Human brain microvessel endothelial cells (hBMVECs) were treated with thrombin (0.1 unit/ml for 30min) in the presence or absence of GSNO (100µM; pretreated for 2hr) or SIN-1 (100µM; pretreated for 2hr) and development of F-actin stress fiber was analyzed by immunofluorescent staining of F-actin bundles by Phalloidin (red-i) and phosphorylated MLC (p-MLC; green-ii). Nuclei were stained by DAPI (blue). B. The resulting digital images were used for quantification of fluorescence and the data is represented by RFU (relative fluorescence unit). C. hBMVECs were cultured on transwell plates and transendothelial electric resistance (TEER) was analyzed. The cells were treated with thrombin (0.1 unit/ml for 5min) in the absence or presence of GSNO (100µM; pretreated for 2hr) or SIN-1 (500µM; pretreated for 2hr). The vertical bars and dotted lines are means of individual data and T-bars are standard deviation. ** p ≤ 0.01 and *** p ≤ 0.001 as compared to the control group. + p ≤ 0.05, ++ p ≤ 0.01, and +++ p ≤ 0.001 as compared to thrombin treated group. All experiments were repeated at least three times.

Article Snippet: Cell culture Primary human brain microvascular endothelial cells (hBMVECs) were purchased from Angio-Proteomie (Cat#: cAP-0002, Atlanta, GA).

Techniques: Staining, Fluorescence, Cell Culture, Standard Deviation, Control

Journal: Toxicology in vitro : an international journal published in association with BIBRA

Article Title: Evaluation of multiwalled carbon nanotube cytotoxicity in cultures of human brain microvascular endothelial cells grown on plastic or basement membrane

doi: 10.1016/j.tiv.2017.03.002

Figure Lengend Snippet: HBMECs were plated on standard tissue culture plastic or matrigel coated plastic and recovered overnight. HBMECs grown on plastic (A) or Matrigel (B) were treated for 24 h with increasing doses of different types of MWCNTs as indicated. Cells were lysed, pelleted, and the supernatant was analyzed for ATP content as a measure of cell viability using the CellTiter-Glo assay. Three independent biological replicates were performed with at least three technical replicates per treatment group. Endpoint analysis shows the groups that are significantly different (*p≤0.05, **p≤0.01) at the highest dose (ANOVA; post-hoc T-Test). In parallel, lysates of similarly treated cells grown on plastic (C) or Matrigel (D) were collected and probed for cleaved PARP, a protein indicative of apoptosis, via western blot analysis. GAPDH was used as a loading control. Lysates from U87 cells treated with 10 μM doxorubicin was used as a positive control. Cells grown on matrigel were isolated using dispase to degrade the matrigel without affecting HBMEC cell integrity. The graph below the western blot displays average fold change of cleaved PARP protein levels relative to GAPDH loading control for three independent biological replicates. Fold change is displayed as the mean ± standard deviation of each experiment.

Article Snippet: Cell Culture Human Brain Microvasculature Endothelial Cells (HBMECs) from Angio-Proteomie were maintained in Endothelial Cell Basal Medium-2 (EBM-2) (Lonza) supplemented with EGM TM -2 SingleQuots® (Lonza) at 37 °C under 5% CO 2 in a humidified incubator.

Techniques: Glo Assay, Western Blot, Control, Positive Control, Isolation, Standard Deviation

Journal: Toxicology in vitro : an international journal published in association with BIBRA

Article Title: Evaluation of multiwalled carbon nanotube cytotoxicity in cultures of human brain microvascular endothelial cells grown on plastic or basement membrane

doi: 10.1016/j.tiv.2017.03.002

Figure Lengend Snippet: HBMECs were plated on plastic to form monolayers and treated the following day with cut uncoated 3H-MWCNT, cut DSPE-PEG coated 3H-MWCNT, or vehicle for the specified times. (A) Cells were washed twice in ice cold PBS, harvested in lysis buffer, and 3H activity was assessed using a scintillation counter. Percent uptake was calculated based on input decays per minute. Three samples were used for each condition and the data are displayed as the mean ± standard error of each measurement. (B) Images taken after 24 h treatment show increased aggregation and sedimentation of uncoated MWCNTs on to HBMECs. By comparison, no aggregated or sedimented cut, DSPE-PEG coated MWCNTs are apparent. *p<0.05 (T-Test).

Article Snippet: Cell Culture Human Brain Microvasculature Endothelial Cells (HBMECs) from Angio-Proteomie were maintained in Endothelial Cell Basal Medium-2 (EBM-2) (Lonza) supplemented with EGM TM -2 SingleQuots® (Lonza) at 37 °C under 5% CO 2 in a humidified incubator.

Techniques: Lysis, Activity Assay, Sedimentation, Comparison

Journal: Toxicology in vitro : an international journal published in association with BIBRA

Article Title: Evaluation of multiwalled carbon nanotube cytotoxicity in cultures of human brain microvascular endothelial cells grown on plastic or basement membrane

doi: 10.1016/j.tiv.2017.03.002

Figure Lengend Snippet: HBMECs were treated in monolayer overnight with 25 μg/mL of each type of MWCNTs and then were replated on matrigel to induce tube formation. After 6 h, photomicrographs of tube formation were taken. Representative images are shown in (A). The average number of HBMEC vessel rings was quantified in three independent biological replicates, with each biological replicate containing triplicate technical replicates. Data are displayed as the mean ± standard error of each experiment (B). Significant differences among groups are indicated: *p<0.05 (ANOVA; post-hoc T-Test).

Article Snippet: Cell Culture Human Brain Microvasculature Endothelial Cells (HBMECs) from Angio-Proteomie were maintained in Endothelial Cell Basal Medium-2 (EBM-2) (Lonza) supplemented with EGM TM -2 SingleQuots® (Lonza) at 37 °C under 5% CO 2 in a humidified incubator.

Techniques:

Journal: Toxicology in vitro : an international journal published in association with BIBRA

Article Title: Evaluation of multiwalled carbon nanotube cytotoxicity in cultures of human brain microvascular endothelial cells grown on plastic or basement membrane

doi: 10.1016/j.tiv.2017.03.002

Figure Lengend Snippet: HBMECs were plated in triplicate on matrigel and allowed to form tube structures, followed by treatment with 25 μg/mL of each type of MWCNT. After 6 h and 16 h, photomicrographs of tube formation were taken. Representative images are shown in (A) for a single biological replicate. The average number of HBMEC vessel rings was quantified and measured in three independent biological replicates, with each biological replicate containing triplicate technical replicates. Data are displayed as the mean ± standard deviation of each experiment (B and C). No significant (p>0.05) difference among groups was detected (ANOVA).

Article Snippet: Cell Culture Human Brain Microvasculature Endothelial Cells (HBMECs) from Angio-Proteomie were maintained in Endothelial Cell Basal Medium-2 (EBM-2) (Lonza) supplemented with EGM TM -2 SingleQuots® (Lonza) at 37 °C under 5% CO 2 in a humidified incubator.

Techniques: Standard Deviation