Hbmec

Manufactured by Merck Group

Sourced in Switzerland

HBMECs are primary human brain microvascular endothelial cells derived from healthy adult donors. They are used for in vitro studies of the blood-brain barrier and related research.

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7 protocols using hbmec

Modulating HBMEC Responses to Ischemia

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HBMEC were purchased from ScienCell and grown to subconfluence in its specialised media (containing 10% FBS) before exposure to OGD (94.5% N2, 0.5% O2 and 5% CO2) or normoxia (75% N2, 20% O2 and 5% CO2) for 0.5 to 4 hours. These OGD conditions were used to mimic a severe ischaemic attack. In some experiments, OGD was followed by 20 hours of reperfusion in which the RPMI media (ischaemic culture medium lacking glucose, pyruvate and foetal bovine serum (FBS), Sigma) was replaced with fresh HBMEC cell media containing 5.5 mM glucose and 10% FBS before exposing cells to normoxic conditions. In other experiments, amiloride (uPA inhibitor, 2.5 µM, Sigma), apocynin (NADPH oxidase inhibitor, 1 mM, Sigma), BAPTA-AM (intracellular calcium chelator, 10 µM, Merck), bisindolylmaleimide (PKC inhibitor, 5 µM, Calbiochem), LY-333531 (PKC-β inhibitor, 1 µM, Enzo Life Sciences), CGP-53353 (PKC-βII inhibitor, 1 µM, Calbiochem) or RO-32-0432 (PKC-α inhibitor, 1 µM, Calbiochem) was also added to culture media during OGD or reperfusion stages. In other experiments, normoxic HBMEC were exposed to phorbol-12myristate-13-acetate (PMA, a PKC activator, 0.1 µM, Sigma) with/out BAPTA-AM.

Rakkar K, & Bayraktutan U. (2016). Increases in intracellular calcium perturb blood-brain barrier via protein kinase C-alpha and apoptosis. Biochimica et biophysica acta, 1862(1).

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Culturing Primary hBMEC and hAEC for Binding Assays

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Primary hBMEC (ScienCell Research laboratories, CA, US) and primary hAEC (ATCC) were cultured in endothelial cell medium (ScienCell Research Laboratories) supplemented with 5% FBS, endothelial cell growth supplement (ScienCell Research Laboratories) and Penicillin (100 U/ml) and streptomycin (100 μg/ml) (ScienCell Research Laboratories). hBMEC were cultured on a Fibronectin matrix (Sigma-Aldrich). For hBMEC passage 5-7 was used, while for hAEC passage 4-7 was used in the binding assays.

Hempel C., Boisen I.M., Efunshile A., Kurtzhals J.A, & Staalsø T. (2015). An automated method for determining the cytoadhesion of Plasmodium falciparum-infected erythrocytes to immobilized cells. Malaria Journal, 14, 112.

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Transendothelial Migration Assay

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Human brain microvascular endothelial cells (HBMECs, Cell Systems, Kirkland, WA, USA) were cultured in complete endothelial cell medium (Cell Systems) and used for experiments up to passage 9. Cells grown to 80% confluency in 24-well plates were used for the coculture assay. On the day of experiment, the medium was changed to complete recombinant serum free medium with 10% exosome free FBS (Cell Systems). Nonstimulated (NS) or activated monocytes were stained with calcein AM (1.5 μM) (Thermo Fisher Scientific) for 30 min at 37 °C, washed with PBS and re-suspended in RPMI containing 10% exosome-free FBS. They were seeded at a concentration of 4 × 10⁵ cells per FluoroBlok^TM insert of 8 μm pore size (Corning, Corning, Coring, NY, USA). The inserts were placed on top of the confluent HBMECs and exosome inhibitor GW4869 (10 μM)^{44 (link)} (Sigma-Aldrich) was immediately added in each insert per stimulation group (NS, IFNα, LPS, I/L). The monocyte migration toward HBMECs was quantified by measuring the increase in fluorescence at 485 nm excitation and 538 nm emission rate in the bottom chamber after 3 h of incubation at 37 °C.

Dalvi P., Sun B., Tang N, & Pulliam L. (2017). Immune activated monocyte exosomes alter microRNAs in brain endothelial cells and initiate an inflammatory response through the TLR4/MyD88 pathway. Scientific Reports, 7, 9954.

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Establishing hBMEC, HEK293T, and THP-1 Cell Cultures

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hBMECs were purchased from ScienCell Research Laboratories (Catalog #1000). HEK293T cells (ATCC® CRL-3216TM) and THP-1 cells (ATCC® TIB-202™) were purchased from American Type Culture Collection. The hBMECs and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, No. c1995500bt) supplemented with 10% fetal bovine serum (Gibco, No.10091148); the THP-1 cells were cultured in RPMI 1640 (Gibco, No. c11875500bt) medium with 10% fetal bovine serum (Gibco, No.10091148). To differentiate THP-1 cells into MΦs, cells were stimulated with 5 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma Aldrich, 79346-1MG) in RPMI complete medium for 72 h and rested in PMA-free RPMI-1640 medium without FBS for an additional 48 h. Further details regarding cell survival assay, microscopy observation, construction of derivative hBMECs, protocol of inhibitor usage, CRISPR screen, and endothelium monolayer model construction assay are provided in SI Appendix, Materials and Methods.

Pan F., Zhu M., Liang Y., Yuan C., Zhang Y., Wang Y., Fan H., Waldor M.K, & Ma Z. (2023). Membrane vesicle delivery of a streptococcal M protein disrupts the blood–brain barrier by inducing autophagic endothelial cell death. Proceedings of the National Academy of Sciences of the United States of America, 120(24), e2219435120.

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Culturing Mouse and Human Brain Endothelial Cells

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Immortalized mouse BECs of the bEnd3 cell line were obtained from the Shanghai Bioleaf Biotech Co., Ltd. Primary human brain microvascular endothelial cells (HBMECs) were purchased from Cell Systems (Kirkland, WA, USA). Cells (bEnd3 and HBMECs) were grown in six-well plates pre-coated with type I or IV collagen (10 μg/mL, Sigma, for 2 h at 37 °C). The culture medium was endothelial basal medium (EBM-2) (Lonza, CC-3156) supplemented with 10% FBS (Gibco), ascorbic acid, L-glutamine, penicillin/streptomycin, and human basic fibroblast growth factor (bFGF) (all from Sigma). Cells were maintained in a humidified incubator at 37 °C and 5% CO2, and the medium was changed every 48 h.

Pang D., Wang L., Dong J., Lai X., Huang Q., Milner R, & Li L. (2018). Integrin α5β1-Ang1/Tie2 receptor cross-talk regulates brain endothelial cell responses following cerebral ischemia. Experimental & Molecular Medicine, 50(9), 117.

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Primary HBMEC Isolation and Culture

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Primary human brain microvascular endothelial cells (HBMECs) from ScienCell™ Research laboratories (Carlsbad, CA, USA, Catalog #1000) were used in passages 3–6 in all assays. Cells were maintained in complete Endothelial Cell Medium (ECM, ScienCell™ Research Laboratories) in a CO₂ incubator at 37 °C, 5% CO₂, and 95% humidity. Before seeding, all flasks and plates were coated in 2 μg/cm² fibronectin bovine plasma (Sigma-Aldrich, St. Louis, MO, USA) and in Mg²⁺- and Ca²⁺-free dPBS. For testing, HBMECs were grown in T75 flasks, detached using Accutase (Sigma-Aldrich), and resuspended in 1 mL glucose-depleted DMEM (Glc(-) DMEM) (Lonza, Basel, Switzerland) before being seeded in the appropriate test plates.

Gu T., Just J., Stenz K.T., Yan Y., Sieljacks P., Wang J., Groennebaek T.S., Jakobsgaard J.E., Rindom E., Herskind J., Gravholt A., Lassen T.R., Jørgensen M., Bæk R., Gutiérrez-Jiménez E., Iversen N.K., Rasmussen P.M., Nyengaard J.R., Jørgensen M.M., de Paoli F., Bøtker H.E., Kjems J., Vissing K, & Drasbek K.R. (2022). The Role of Plasma Extracellular Vesicles in Remote Ischemic Conditioning and Exercise-Induced Ischemic Tolerance. International Journal of Molecular Sciences, 23(6), 3334.

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Modeling Amyloid-β Induced Senescence in HBMECs

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The human brain microvascular endothelial cells (HBMECs, #ACBRI 376), attachment factor and cell culture media were purchased from the Cell Systems (Kirkland, WA, U.S). HBMECs at an early passage (p4) with cell confluency upto ~70–80% were used for all experiments. Aβ1–42 oligomers were prepared and characterized as previously described (32 (link)–35 (link)) and were a kind gift from Dr. Rosenberry (Mayo Clinic, Florida). For Aβ1–42 oligomer-induced senescence experiments, HBMECs were treated with 5 μM Aβ1–42 oligomers and incubated in a humidified incubator containing 5% CO2 maintained at 37°C for seven days. For Rac 1 inhibition studies, HBMECs were cultured with Rac 1 inhibitor (NSC 23766, Sigma Aldrich) overnight in a humidified incubator containing 5% CO₂ maintained at 37°C.

Kulkarni T., Angom R.S., Das P., Bhattacharya S, & Mukhopadhyay D. (2019). Nanomechanical insights: Amyloid beta oligomer-induced senescent brain endothelial cells. Biochimica et biophysica acta. Biomembranes, 1861(12), 183061.

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