human brain endothelial cells hbec5i  (ATCC)

Journal: Antioxidants (Basel, Switzerland)

Article Title: Protective Roles of Zinc and Selenium Against Oxidative Stress in Brain Endothelial Cells Under Shear Stress.

doi: 10.3390/antiox14040451

Figure Lengend Snippet: Figure 2. ZO1 localization in HBEC5i under different flow conditions. Human brain endothelial cells (HBEC5i) were cultured in the LiveBox2 system and subjected to a controlled flow rate ranging from 0 µL/min (no flow) to 200 µL/min. In the absence of flow (A), ZO1 (red) was properly localized at the cell periphery, ensuring tight junction integrity. Under shear stress conditions (200 µL/min, (B)), ZO1 exhibited an altered distribution, appearing stretched and disorganized. Nuclei were counterstained with DAPI (blue). The comparison of data distribution for each flow condition is reported as a violin plot (C). Control (no flow) 3.87 ± 0.16, 200 µL/min (flow) 1.55 ± 0.16, p < 0.05. Quantitative analysis revealed that the membrane/cytoplasm fluorescence intensity ratio of ZO1 decreased by approximately 60% in shear-stressed cells compared to controls (p < 0.05). The experiment was conducted three times, with five fields captured per experimental condition using a DCF350FX camera. Total magnification: 200×. Scale bar: 50 µm.

Article Snippet: Human brain endothelial cells (HBEC5i) were obtained from ATCC (Milan, Italy), and cultured in a 1:1 mixture of DMEM/HAM’s F12, supplemented with 10% FBS (Fetal Bovine Serum), 1% antibiotics (penicillin and streptomycin, Euroclone, Milan, Italy), and EGM (Endothelial Growth Medium (% for 500 mL), consisting of hydrocortisone (0.02% 0.2 mL), bFGF (2% 2 mL), VEGF (Vascular Endothelial Growth Factor, 0.1% 0.5 mL), R3-IGF1 (Insulinlike Growth Factor 1, 0.1% 0.5 mL), ascorbic acid (0.1% 0.5 mL), hEGF (human Endothelial Growth Factor, 0.1% 0.5 mL), and GA-1000 (gentamicin sulfate–amphotericin, 0.1% 0.5 mL), and heparin (0.1% 0.5 mL) (Lonza, Euroclone, Milan, Italy), in a 5% CO2 humidified atmosphere at 37 ◦C.

Techniques: Cell Culture, Shear, Comparison, Control, Membrane, Fluorescence

Journal: Antioxidants (Basel, Switzerland)

Article Title: Protective Roles of Zinc and Selenium Against Oxidative Stress in Brain Endothelial Cells Under Shear Stress.

doi: 10.3390/antiox14040451

Figure Lengend Snippet: Figure 3. NRF2 nuclear translocation in HBEC5i cells under shear stress conditions. Human brain endothelial cells (HBEC5i) were cultured in the LiveBox2 system, which allows for a precise regu- lation of flow rates from 0 µL/min (no flow) to 200 µL/min. In (A), cells maintained under static conditions (0 µL/min) exhibited cytoplasmic NRF2 localization (green), as confirmed by the plot profile (DAPI-nucleus: blue line; NRF2 signal: green line). In contrast, exposure to a flow rate of 200 µL/min (B) induced NRF2 nuclear translocation, as illustrated by the corresponding plot profile. The comparison of data distribution for each flow condition is reported as a violin plot (C). Quantita- tive analysis revealed that the percentage of cells exhibiting NRF2 nuclear localization increased from 31.86% ± 1.34 under static conditions to 74.53% ± 2.76 under shear stress (p < 0.05). Nuclei were counterstained with DAPI (blue). The experiments were conducted three times, capturing five fields for each experimental condition using a DCF350FX camera. Total magnification: 200×. Scale bar: 50 µm.

Article Snippet: Human brain endothelial cells (HBEC5i) were obtained from ATCC (Milan, Italy), and cultured in a 1:1 mixture of DMEM/HAM’s F12, supplemented with 10% FBS (Fetal Bovine Serum), 1% antibiotics (penicillin and streptomycin, Euroclone, Milan, Italy), and EGM (Endothelial Growth Medium (% for 500 mL), consisting of hydrocortisone (0.02% 0.2 mL), bFGF (2% 2 mL), VEGF (Vascular Endothelial Growth Factor, 0.1% 0.5 mL), R3-IGF1 (Insulinlike Growth Factor 1, 0.1% 0.5 mL), ascorbic acid (0.1% 0.5 mL), hEGF (human Endothelial Growth Factor, 0.1% 0.5 mL), and GA-1000 (gentamicin sulfate–amphotericin, 0.1% 0.5 mL), and heparin (0.1% 0.5 mL) (Lonza, Euroclone, Milan, Italy), in a 5% CO2 humidified atmosphere at 37 ◦C.

Techniques: Translocation Assay, Shear, Cell Culture, Comparison

Journal: Antioxidants (Basel, Switzerland)

Article Title: Protective Roles of Zinc and Selenium Against Oxidative Stress in Brain Endothelial Cells Under Shear Stress.

doi: 10.3390/antiox14040451

Figure Lengend Snippet: Figure 5. HBEC5i cells were subjected to a flow rate of 200 µL/min in the presence of 10 µM ZnCl2. As shown in (A), NRF2 (green) remained localized in the cytoplasm, indicating that oxidative stress was mitigated. The corresponding graph in (B) illustrates a profile comparison between cells exposed to shear stress with and without ZnCl2 treatment, revealing a statistically significant reduction in NRF2 nuclear translocation in ZnCl2-treated cells. Nuclei were counterstained with DAPI (blue). The comparison of data distribution for each flow condition is reported as a violin plot (C). ZnCl2 40.08% ± 2.19, 200 µL/min (flow) 69.45% ± 2.03, p < 0.05. The experiments were performed three times, capturing five fields per experimental condition using a DCF350FX camera. Total magnification: 200×. Scale bar: 50 µm.

Article Snippet: Human brain endothelial cells (HBEC5i) were obtained from ATCC (Milan, Italy), and cultured in a 1:1 mixture of DMEM/HAM’s F12, supplemented with 10% FBS (Fetal Bovine Serum), 1% antibiotics (penicillin and streptomycin, Euroclone, Milan, Italy), and EGM (Endothelial Growth Medium (% for 500 mL), consisting of hydrocortisone (0.02% 0.2 mL), bFGF (2% 2 mL), VEGF (Vascular Endothelial Growth Factor, 0.1% 0.5 mL), R3-IGF1 (Insulinlike Growth Factor 1, 0.1% 0.5 mL), ascorbic acid (0.1% 0.5 mL), hEGF (human Endothelial Growth Factor, 0.1% 0.5 mL), and GA-1000 (gentamicin sulfate–amphotericin, 0.1% 0.5 mL), and heparin (0.1% 0.5 mL) (Lonza, Euroclone, Milan, Italy), in a 5% CO2 humidified atmosphere at 37 ◦C.

Techniques: Comparison, Shear, Translocation Assay

Journal: Antioxidants (Basel, Switzerland)

Article Title: Protective Roles of Zinc and Selenium Against Oxidative Stress in Brain Endothelial Cells Under Shear Stress.

doi: 10.3390/antiox14040451

Figure Lengend Snippet: Figure 6. ZnCl2 (10 µM) maintains proper ZO-1 under shear stress conditions. HBEC5i cells were exposed to a 200 µL/min flow rate in the presence of 10 µM ZnCl2. In (A), ZO1 protein (red) remained correctly localized at the cell periphery, preserving tight junction integrity, whereas in (B), cells exposed to shear stress without ZnCl2 exhibited disrupted ZO-1 localization. The comparison of data distribution for each flow condition is reported as a violin plot (C). ZnCl2 2.37 ± 0.15, 200 µL/min (flow) 1.34 ± 0.08, p < 0.05. Quantitative analysis revealed a statistically significant increase in the membrane/cytoplasm fluorescence intensity ratio of ZO-1 in ZnCl2-treated cells compared to untreated cells. Nuclei were counterstained with DAPI (blue). The experiments were performed three times, capturing five fields per experimental condition using DCF350FX camera. Total magnification: 200×. Scale bar: 50 µm.

Article Snippet: Human brain endothelial cells (HBEC5i) were obtained from ATCC (Milan, Italy), and cultured in a 1:1 mixture of DMEM/HAM’s F12, supplemented with 10% FBS (Fetal Bovine Serum), 1% antibiotics (penicillin and streptomycin, Euroclone, Milan, Italy), and EGM (Endothelial Growth Medium (% for 500 mL), consisting of hydrocortisone (0.02% 0.2 mL), bFGF (2% 2 mL), VEGF (Vascular Endothelial Growth Factor, 0.1% 0.5 mL), R3-IGF1 (Insulinlike Growth Factor 1, 0.1% 0.5 mL), ascorbic acid (0.1% 0.5 mL), hEGF (human Endothelial Growth Factor, 0.1% 0.5 mL), and GA-1000 (gentamicin sulfate–amphotericin, 0.1% 0.5 mL), and heparin (0.1% 0.5 mL) (Lonza, Euroclone, Milan, Italy), in a 5% CO2 humidified atmosphere at 37 ◦C.

Techniques: Shear, Preserving, Comparison, Membrane, Fluorescence

Journal: Antioxidants (Basel, Switzerland)

Article Title: Protective Roles of Zinc and Selenium Against Oxidative Stress in Brain Endothelial Cells Under Shear Stress.

doi: 10.3390/antiox14040451

Figure Lengend Snippet: Figure 7. Na2SeO3 (100 nM) fails to prevent NRF2 nuclear translocation under shear stress. HBEC5i cells were subjected to a 200 µL/min flow rate in the presence of 100 nM Na2SeO3 (A). NRF2 (green) translocated to the nucleus, indicating oxidative stress induction, similar to cells exposed to shear stress without Na2SeO3 (B). The comparison of data distribution for each flow condition is reported as a violin plot (C). Na2SeO3 71.08% ± 2.35, 200 µL/min (flow) 73.47% ± 2.37, not significant. Quantitative analysis revealed no significant difference in the percentage of cells exhibiting NRF2 nuclear translocation between Na2SeO3-treated and untreated shear-stressed cells. Nuclei were counterstained with DAPI (blue). The experiments were conducted three times, capturing five fields per experimental condition using a DCF350FX camera. Total magnification: 200×. Scale bar: 50 µm.

Article Snippet: Human brain endothelial cells (HBEC5i) were obtained from ATCC (Milan, Italy), and cultured in a 1:1 mixture of DMEM/HAM’s F12, supplemented with 10% FBS (Fetal Bovine Serum), 1% antibiotics (penicillin and streptomycin, Euroclone, Milan, Italy), and EGM (Endothelial Growth Medium (% for 500 mL), consisting of hydrocortisone (0.02% 0.2 mL), bFGF (2% 2 mL), VEGF (Vascular Endothelial Growth Factor, 0.1% 0.5 mL), R3-IGF1 (Insulinlike Growth Factor 1, 0.1% 0.5 mL), ascorbic acid (0.1% 0.5 mL), hEGF (human Endothelial Growth Factor, 0.1% 0.5 mL), and GA-1000 (gentamicin sulfate–amphotericin, 0.1% 0.5 mL), and heparin (0.1% 0.5 mL) (Lonza, Euroclone, Milan, Italy), in a 5% CO2 humidified atmosphere at 37 ◦C.

Techniques: Translocation Assay, Shear, Comparison

Journal: Antioxidants (Basel, Switzerland)

Article Title: Protective Roles of Zinc and Selenium Against Oxidative Stress in Brain Endothelial Cells Under Shear Stress.

doi: 10.3390/antiox14040451

Figure Lengend Snippet: Figure 8. Na2SeO3 (100 nM) treatment does not maintain ZO-1 localization under shear stress conditions. HBEC5i cells were subjected to a 200 µL/min flow rate in the presence of 100 nM Na2SeO3. In (A), ZO-1 (red) failed to maintain its perimetral localization, mirroring cells exposed to shear stress without Na2SeO3 (B). The comparison of data distribution for each flow condition is reported as a violin plot (C). Na2SeO3 1.45 ± 0.08, 200 µL/min (flow) 1.30 ± 0.02, not significant. Quantitative analysis confirmed that there was no significant difference in the membrane/cytoplasm fluorescence intensity ratio of ZO-1 between Na2SeO3-treated and untreated shear-stressed cells. Nuclei were counterstained with DAPI (blue). The experiments were performed three times, capturing five fields per experimental condition using a DCF350FX camera. Total magnification: 200×. Scale bar: 50 µm.

Article Snippet: Human brain endothelial cells (HBEC5i) were obtained from ATCC (Milan, Italy), and cultured in a 1:1 mixture of DMEM/HAM’s F12, supplemented with 10% FBS (Fetal Bovine Serum), 1% antibiotics (penicillin and streptomycin, Euroclone, Milan, Italy), and EGM (Endothelial Growth Medium (% for 500 mL), consisting of hydrocortisone (0.02% 0.2 mL), bFGF (2% 2 mL), VEGF (Vascular Endothelial Growth Factor, 0.1% 0.5 mL), R3-IGF1 (Insulinlike Growth Factor 1, 0.1% 0.5 mL), ascorbic acid (0.1% 0.5 mL), hEGF (human Endothelial Growth Factor, 0.1% 0.5 mL), and GA-1000 (gentamicin sulfate–amphotericin, 0.1% 0.5 mL), and heparin (0.1% 0.5 mL) (Lonza, Euroclone, Milan, Italy), in a 5% CO2 humidified atmosphere at 37 ◦C.

Techniques: Shear, Comparison, Membrane, Fluorescence

Journal: Bioengineering

Article Title: A Versatile Microfluidic Device System that Lacks a Synthetic Extracellular Matrix Recapitulates the Blood–Brain Barrier and Dynamic Tumor Cell Interaction

doi: 10.3390/bioengineering11101008

Figure Lengend Snippet: The growth kinetics of the HBEC5i (endothelial cells), U87MG (glioblastoma tumoral cells), and astrocyte cell lines. The top central graphs show the quantification of the growth rate with representative micrographs of the time course of proliferation in the culture plate below each graph. The scale bar represents 100 µm in all.

Article Snippet: Human U87MG cells; glioblastoma multiforme from the American Type Culture Collection (ATCC, Manassas, VA, USA), with EMEM +10% fetal bovine serum [ ], Merck/Millipore Sigma Burlington, MA, USA), HBEC5i (from the ATCC, with DMEM/F12 +10% fetal bovine serum, made in Manassas, Virginia USA), and astrocytes from ScienCell, Carlsbad, CA, USA (catalog number 1800, with supplement culture medium 1801) were utilized, following the specifications described in the technical datasheet, All cell lines were maintained at 80% confluence for cell subculturing, and all experiments were conducted with subcultures of fewer than 8 subcultures. shows the general process carried out in this study.

Techniques:

Journal: Bioengineering

Article Title: A Versatile Microfluidic Device System that Lacks a Synthetic Extracellular Matrix Recapitulates the Blood–Brain Barrier and Dynamic Tumor Cell Interaction

doi: 10.3390/bioengineering11101008

Figure Lengend Snippet: The cell migration ability of ( A ) U87MG endothelial cells and ( B ) HBEC5i at different starting cell densities. Migrating cells are indicated in yellow.

Article Snippet: Human U87MG cells; glioblastoma multiforme from the American Type Culture Collection (ATCC, Manassas, VA, USA), with EMEM +10% fetal bovine serum [ ], Merck/Millipore Sigma Burlington, MA, USA), HBEC5i (from the ATCC, with DMEM/F12 +10% fetal bovine serum, made in Manassas, Virginia USA), and astrocytes from ScienCell, Carlsbad, CA, USA (catalog number 1800, with supplement culture medium 1801) were utilized, following the specifications described in the technical datasheet, All cell lines were maintained at 80% confluence for cell subculturing, and all experiments were conducted with subcultures of fewer than 8 subcultures. shows the general process carried out in this study.

Techniques: Migration

Journal: Bioengineering

Article Title: A Versatile Microfluidic Device System that Lacks a Synthetic Extracellular Matrix Recapitulates the Blood–Brain Barrier and Dynamic Tumor Cell Interaction

doi: 10.3390/bioengineering11101008

Figure Lengend Snippet: Co-culture of U87MG-HBEC5i acquired by confocal microscopy: the cell migration process. The cellular cytoskeleton is shown with the expression of phalloidin (green), and the nuclei were assessed with DAPI (blue). Cell–cell interactions are shown with yellow arrows, which occurred through interconnecting channels. The scale bar represents 100 µm.

Article Snippet: Human U87MG cells; glioblastoma multiforme from the American Type Culture Collection (ATCC, Manassas, VA, USA), with EMEM +10% fetal bovine serum [ ], Merck/Millipore Sigma Burlington, MA, USA), HBEC5i (from the ATCC, with DMEM/F12 +10% fetal bovine serum, made in Manassas, Virginia USA), and astrocytes from ScienCell, Carlsbad, CA, USA (catalog number 1800, with supplement culture medium 1801) were utilized, following the specifications described in the technical datasheet, All cell lines were maintained at 80% confluence for cell subculturing, and all experiments were conducted with subcultures of fewer than 8 subcultures. shows the general process carried out in this study.

Techniques: Co-Culture Assay, Confocal Microscopy, Migration, Expressing

Journal: Bioengineering

Article Title: A Versatile Microfluidic Device System that Lacks a Synthetic Extracellular Matrix Recapitulates the Blood–Brain Barrier and Dynamic Tumor Cell Interaction

doi: 10.3390/bioengineering11101008

Figure Lengend Snippet: The culture evolution at different time points and the process of cell invasion. Glioblastoma U87MG cells are shown in green, endothelial HBEC5i cells are shown in red, cell nuclei are shown in blue (DAPI), and cell–cell interactions through interconnecting channels are shown by green and red arrows. The expression of cytokines was carried out by combining the triplicate model to obtain the necessary amount of culture medium (cytokine sample = 1). The scale bar represents 200 µm.

Article Snippet: Human U87MG cells; glioblastoma multiforme from the American Type Culture Collection (ATCC, Manassas, VA, USA), with EMEM +10% fetal bovine serum [ ], Merck/Millipore Sigma Burlington, MA, USA), HBEC5i (from the ATCC, with DMEM/F12 +10% fetal bovine serum, made in Manassas, Virginia USA), and astrocytes from ScienCell, Carlsbad, CA, USA (catalog number 1800, with supplement culture medium 1801) were utilized, following the specifications described in the technical datasheet, All cell lines were maintained at 80% confluence for cell subculturing, and all experiments were conducted with subcultures of fewer than 8 subcultures. shows the general process carried out in this study.

Techniques: Expressing