bend3 murine brain endothelial cells  (ATCC)

Journal: ACS Applied Bio Materials

Article Title: Real-Time Monitoring of the Cytotoxic Effect of Oxygen-Sensitive Fluorescent Poly(styrene-maleic anhydride) Nanoparticles Using Electrical-Substrate Impedance Sensing

doi: 10.1021/acsabm.5c01443

Figure Lengend Snippet: Effects of SMA NPs on viable cell density and toxicity of NIH 3T3 fibroblasts and bEnd3 brain endothelial cells assessed by alamarBlue assay: (a–c) NIH 3T3 fibroblasts; (d–f) bEnd3 cells. (a) Fluorescence intensity of reduced resazurin by NIH 3T3 fibroblasts on day 5 of culture corresponding to 3 days of incubation with SMA NPs. (b) Relative cell density of NIH 3T3 fibroblasts on day 5. (c) Cytotoxicity of SMA NPs on NIH 3T3 fibroblasts. (d) Fluorescence intensity of reduced resazurin by bEnd3 cells on day 5 of culture corresponding to 3 days of incubation with SMA NPs. (e) Relative cell density of bEnd3 cells on day 5. (f) Cytotoxicity of SMA NPs on bEnd3 cells. In all panels, the fluorescence intensity is compared to the negative control (brown bars). **** p < 0.0001. n = 4.

Article Snippet: bEnd3 murine brain endothelial cells, obtained from the American Type Culture Collection (ATCC, CRL-2299, VA), were cultured in Gibco DMEM (low glucose) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 0.1% gentamicin (Thermo Fisher Scientific).

Techniques: Alamar Blue Assay, Fluorescence, Incubation, Negative Control

Journal: ACS Applied Bio Materials

Article Title: Real-Time Monitoring of the Cytotoxic Effect of Oxygen-Sensitive Fluorescent Poly(styrene-maleic anhydride) Nanoparticles Using Electrical-Substrate Impedance Sensing

doi: 10.1021/acsabm.5c01443

Figure Lengend Snippet: ECIS measurements of cellular response to the introduction of fluorescent SMA NPs. (a) Normalized five-day real-time ECIS measurement of NIH 3T3 fibroblasts, showing real-time data of cell attachment, proliferation, and response to fluorescent SMA NPs at varying concentrations. Capacitance was recorded at 64 kHz. The dotted blue line indicates the time when the fluorescent SMA NPs were added to each well. (b) Capacitance values from NIH 3T3 cells on day five at 110 h in culture. (c) Cytotoxicity percentage of NIH 3T3 fibroblasts in response to contact with SMA NPs. (d) Normalized five-day real-time ECIS measurement of bEnd3brain epithelial cells, showing real-time data of attachment, proliferation, and response to fluorescent SMA NPs. Capacitance was recorded at 64 kHz. The dotted blue line indicates the time when the fluorescent SMA NPs were added to each well. (e) Capacitance values of bEnd3 cells on day five at 110 h in culture. (f) Cytotoxicity percentage of brain epithelial bEnd3 cells in response to contact with SMA NPs. In panels b, c, e, and f, the capacitances and cytotoxicities are compared to the negative control (brown bars). **** p < 0.0001. n = 4.

Article Snippet: bEnd3 murine brain endothelial cells, obtained from the American Type Culture Collection (ATCC, CRL-2299, VA), were cultured in Gibco DMEM (low glucose) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 0.1% gentamicin (Thermo Fisher Scientific).

Techniques: Cell Attachment Assay, Negative Control

Journal: ACS Applied Bio Materials

Article Title: Real-Time Monitoring of the Cytotoxic Effect of Oxygen-Sensitive Fluorescent Poly(styrene-maleic anhydride) Nanoparticles Using Electrical-Substrate Impedance Sensing

doi: 10.1021/acsabm.5c01443

Figure Lengend Snippet: Barrier condition analysis of bENd3 brain epithelial cells after addition of SMA NPs at different concentrations. (a) Real-time ECIS resistance measurements at 4 kHz. (b) Comparison of resistance values between different experimental conditions, before adding SMA NPs on day 4, after SMA NPs addition on day 8, and after media replacement on day 10. (c) Real-time ECIS capacitance measurements at 64 kHz. (d) Comparison of normalized capacitance between different experimental conditions, before adding SMA NPs, after SMA NPs addition, and after media replacement. In panels b and d, the capacitance is compared to the negative control (brown bars). ** p < 0.0001. n = 5.

Article Snippet: bEnd3 murine brain endothelial cells, obtained from the American Type Culture Collection (ATCC, CRL-2299, VA), were cultured in Gibco DMEM (low glucose) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 0.1% gentamicin (Thermo Fisher Scientific).

Techniques: Comparison, Negative Control

Journal: ACS Applied Bio Materials

Article Title: Real-Time Monitoring of the Cytotoxic Effect of Oxygen-Sensitive Fluorescent Poly(styrene-maleic anhydride) Nanoparticles Using Electrical-Substrate Impedance Sensing

doi: 10.1021/acsabm.5c01443

Figure Lengend Snippet: Fluorescence-phase contrast micrographs of randomly selected samples. Micrographs show NIH 3T3 fibroblasts (top panel) and bEnd3 brain endothelial cells (bottom panel) containing fluorescent SMA NPs (green) on day 7 at multiple concentrations. Scale bar = 275 μm.

Article Snippet: bEnd3 murine brain endothelial cells, obtained from the American Type Culture Collection (ATCC, CRL-2299, VA), were cultured in Gibco DMEM (low glucose) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 0.1% gentamicin (Thermo Fisher Scientific).

Techniques: Fluorescence

Journal: ACS Applied Bio Materials

Article Title: Real-Time Monitoring of the Cytotoxic Effect of Oxygen-Sensitive Fluorescent Poly(styrene-maleic anhydride) Nanoparticles Using Electrical-Substrate Impedance Sensing

doi: 10.1021/acsabm.5c01443

Figure Lengend Snippet: SEM images showing the cell–NP interaction after 7 days of culture with fluorescent SMA NPs. (Top panel) NIH 3T3 fibroblasts. (Bottom panel) bEnd3 brain endothelial cells. Turquoise hexagons point to SMA NPs attached to cells and intercalated into the extracellular matrix (ECM). The left and right panels are replicate samples. Scale bar = 10 μm. Zoomed micrograph scale bar = 400 nm.

Article Snippet: bEnd3 murine brain endothelial cells, obtained from the American Type Culture Collection (ATCC, CRL-2299, VA), were cultured in Gibco DMEM (low glucose) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 0.1% gentamicin (Thermo Fisher Scientific).

Techniques:

Journal: Nature Communications

Article Title: The liver and muscle secreted HFE2-protein maintains central nervous system blood vessel integrity

doi: 10.1038/s41467-024-45303-1

Figure Lengend Snippet: a Knockout of liver Hfe2 reduces pericyte coverage in section from the cerebral cortex (mean ± s.e.m.; unpaired two-tail t-test; AAV8-GFP n = 3, Hfe2 ΔAlb-cre n = 4, and AAV8-AlbCre n = 3). Scale bar, 50μm. b Quantification through quantitative RT-PCR shows a reduction in PDGF-B mRNA levels in liver knock out animals (mean ± s.e.m.; paired two-tail t-test; Hfe2 fl/fl n = 3 and Hfe2 ΔAlb-cre n = 3). c Quantification of PDGF-B mRNA in cultured bEnd.3 cells following indicated treatments. Treatment with RGMa leads to a significant reduction of PDGF-B mRNA levels that is rescued with HFE2 addition (mean ± s.e.m.; paired two-tail t-test; HFE2 n = 3, RGMa n = 4, and RGMa+ HFE2 n = 5; n representing an independent experiment). d Immunocytochemistry of claudin-5 in human primary endothelial cell monolayer after PBS, HFE2, RGMa, and RGMa+HFE2 treatments and quantification (mean ± s.e.m.; unpaired two-tail t-test; replicates n = 3). Scale bar, 100 µm. e Western blotting of Claudin-5 expression in bEnd3 cell lysates after indicated treatment (mean ± s.e.m.; unpaired two-tailed t-test; replicates n = 3). f Transwell permeability leakage assay performed on a monolayer of bEnd3 cells using HRP (mean ± s.e.m.; paired one-tail t-test; replicates n = 3). g Transwell permeability leakage assay performed on a monolayer of human primary endothelial cells using 70 kDa FITC-dextran (mean ± s.e.m.; paired two-tail t-test; replicates n = 3). h In vitro TEER analysis shows that RGMa alters the integrity of a monolayer of bEnd3 cells, this is rescued by HFE2 (mean ± s.e.m.; unpaired two-tail t-test; replicates n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file (includes exact p-values).

Article Snippet: The endothelial monolayer permeability assay was performed as previously described DiStefano et al. Murine Brain Endothelial cells (bEnd3) (ATCC®CRL-2299 TM ) were seeded on 3 μm pore Transwell inserts (Corning Life Sciences) coated with 10 μg/ml human plasma fibronectin (Sigma, FC010).

Techniques: Knock-Out, Quantitative RT-PCR, Cell Culture, Immunocytochemistry, Western Blot, Expressing, Two Tailed Test, Permeability, In Vitro

Journal: EMBO Molecular Medicine

Article Title: Vulnerability of invasive glioblastoma cells to lysosomal membrane destabilization

doi: 10.15252/emmm.201809034

Figure Lengend Snippet: A MDGI‐silenced (shMDGI1) BT12 and BT13 cells were stained with anti‐galectin‐1 (LGALS1, green) and anti‐LAMP2 (red) antibodies 6 days after silencing. Co‐localization of the LGALS1 with LAMP2 is seen as yellow colour (merge). Scale bar: 10 μm. B Lysosomal extracts of BT12 and BT13 cells were prepared 6 days after MDGI silencing by density gradient ultracentrifugation. Western blot analyses show the presence of lysosomes and purity of the collected fractions (1 + 2 and 3 + 4). Antibodies against LAMP2 and Calnexin‐1 were used for the localization of lysosomes (fractions 1 + 2) and endoplasmic reticulum (fractions 3 + 4), respectively. C Measurement of the viability of murine brain endothelial (bEND3) cells using MTT assay at the indicated clemastine concentrations and time points ( n = 12). A dashed line marks the 50% cell viability. Data are represented as mean ± SD. D Co‐localization of the LGALS1 (green) with the lysosomal marker protein LAMP2 (red) is seen as yellow colour (merge) in clemastine (1 μg/ml)‐treated BT12 cells. Scale bar: 10 μm. Source data are available online for this figure.

Article Snippet: Human glioma cell line U87MG and murine brain microvessel endothelial cells bEND3 (ATCC) were maintained for not more than 10 and 15 passages, respectively, in DMEM containing 1 g/l glucose, and with the same supplements described above.

Techniques: Staining, Western Blot, MTT Assay, Marker

Journal: Journal of Alzheimer's disease : JAD

Article Title: Protective Role of S-Nitrosoglutathione (GSNO) Against Cognitive Impairment in Rat Model of Chronic Cerebral Hypoperfusion

doi: 10.3233/JAD-121786

Figure Lengend Snippet: Anti-inflammatory activity of GSNO. A) The effect of GSNO treatment on the expression of vasoinflammatory markers (ICAM-1 and VCAM-1) in the brains of rats treated with permanent BCCAO was examined by real time quantitative PCR and by immunofluorescence staining of paraffin sections as described in method section. In the quantitative PCR analysis, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was quantified as an internal control. B) Further, to study the mechanism of GSNO in endothelial inflammatory signaling events, the cultured mouse brain endothelial cells (bEND3) were pretreated with GSNO (250 μM) for 3 h and then treated with cytokine mix (Cyto; TNFα, IL1β, and IFNγ; 25 ng/ml each). Following incubation for 0.5 h with cytokine mix, the activities of nuclear factor κ-B (NFκB) were analyzed by western analysis for nuclear p65 and cytoplasmic IκB and by gel-shift assay of NFκB DNA binding activity (i). The activities of signal transducer and activator of transcription 1 (STAT1) and STAT3 were analyzed by western analysis of phospho-(Tyr701)-STAT1 and phospho-(Tyr705)-STAT3 (ii). C) Following GSNO cytokine treatment, the mRNA (i) and protein (ii) levels of ICAM-1 and VCAM-1 were analyzed. In these experiments, GSNO was pretreated for 3 h before treatment with cytokine mix. The mRNA and proteins were extracted 6 h and 12 h after cytokine treatment. The data was analyzed for mean values and standard error for all experiments; *p < 0.05 and **p < 0.01 for comparison to vehicle (VHC) treated control (CTL) group; +p <0.05 and ++p <0.01 for comparison to cytokine (Cyto) treated group.

Article Snippet: Cell culture and drug treatment The bEND3 murine brain endothelial cell line and BV2 murine microglial cell line were purchase from American Type Culture Collection (ATCC) and maintained in Dulbecco's-Modified-Eagles-Medium (DMEM) containing 10% fetal bovine serum, and 1% penicillin/streptomycin under 37°C, 5% CO 2 incubator.

Techniques: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Control, Cell Culture, Incubation, Western Blot, Gel Shift, Binding Assay, Comparison

Journal: Journal of Alzheimer's disease : JAD

Article Title: Protective Role of S-Nitrosoglutathione (GSNO) Against Cognitive Impairment in Rat Model of Chronic Cerebral Hypoperfusion

doi: 10.3233/JAD-121786

Figure Lengend Snippet: Promotion of Aβ uptake by GSNO in endothelial and microglial cells. A) To assess the effect of GSNO on dynamin-2-mediated endothelial Aβ uptake, the mammalian expression vector expressing human dynamin-2 was transfected to bEND3 murine brain endothelial cells, and then the cellular levels of dynamin-2 and the endothelial uptake of fluorescence-labeled Aβ (FAM-Aβ) were analyzed (i). To examine whether GSNO is able to S-nitrosylate dynamin-2, the bEND3 cells were treated with GSNO (250 μM/3 h) and subjected to biotin-switch assay. The resulting S-biotinylated (S-nitrosylated) proteins in whole cell lysates were detected by western analysis using antibody specific to biotin (ii). To assess the involvement of S-nitrosylation mechanism on GSNO-induced Aβ uptake, the wild type, C86A mutant, or C607A mutant dynamin-2 was transfected to bEND3 cells and biotin-switch assay and Aβ uptake assay were performed following the GSNO treatment (250 μM/3 h). Following the pull-down of S-biotinylated proteins with avidin-conjugated agarose beads, the levels of co-precipitated dynamin-2 (DNM-2) were analyzed by western blot. The levels of dynamin-2 in whole cell lysates were analyzed to examine the total dynamin-2 levels. The levels of β-actin in whole cell lysates were used for internal loading control. B) To examine the effect of GSNO on Aβ uptake, FAM-Aβ uptake assay was performed in bEND3 and BV2 microglial cells. Before the uptake assay, FAM-Aβ peptides were preincubated with basal media (fresh DMEM) or astroglial conditioned media (CM), which included lipidated ApoE (see Supplementary Data 1). To examine whether CM-mediated Aβ uptake is mediated through LRP1, the Aβ uptake assay was performed following the treatment of cells with LRP1 inhibitor, receptor-associated protein (Rap) (i). To examine whether GSNO enhances basal media (BM) or CM-mediated Aβ uptake, the cells were pretreated with GSNO (250 μM/3 hrs) prior to performance of Aβ uptake assay (ii). To examine whether GSNO treatment enhances the endothelial uptake of Aβ40 as well as Aβ42, the bEND3 cells were incubated with GSNO and then subjected to Aβ uptake assay using FAM-Aβ40 and FAM-Aβ42 peptides which were pre-incubated with CM (iii). Aβ uptake was also visualized under a fluorescent microscope (iv). C) In addition to bEND3 cells, the Aβ uptake assay was also performed on BV2 microglia under the same experimental conditions. The data were analyzed for mean values and standard error for all experiments; *p < 0.05, **p < 0.01, and ***p < 0.001 for comparison to mock control (empty), BM, or vehicle (VHC) treated group; +p < 0.05 for comparison to CM treated group.

Article Snippet: Cell culture and drug treatment The bEND3 murine brain endothelial cell line and BV2 murine microglial cell line were purchase from American Type Culture Collection (ATCC) and maintained in Dulbecco's-Modified-Eagles-Medium (DMEM) containing 10% fetal bovine serum, and 1% penicillin/streptomycin under 37°C, 5% CO 2 incubator.

Techniques: Expressing, Plasmid Preparation, Transfection, Fluorescence, Labeling, Biotin Switch Assay, Western Blot, Mutagenesis, Avidin-Biotin Assay, Control, Incubation, Microscopy, Comparison