Immortalized mouse brain endothelial cells (bEnd3) from ATCC (Manassas, VA, USA) were used as a representative model for a mouse BBB endothelium. bEnd3, passage 25–35, were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin G (100 units/mL) and streptomycin (100 μg/mL); all were purchased from ATCC (Manassas, VA, USA). Cultures were maintained in a humidified atmosphere (5% CO2) at 37 °C incubator (VWR International, Radnor, PA, USA).
Bend 3
Manufactured by American Type Culture Collection
Sourced in United States, United Kingdom
The BEnd.3 is a cell line derived from mouse brain endothelial cells. It is a commonly used in vitro model for studying blood-brain barrier properties and functions.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (18 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (17 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (10 mentions)
Fetal bovine serum (fbs), by Merck Group (9 mentions)
Penicillin, by Merck Group (7 mentions)
Streptomycin, by Merck Group (7 mentions)
C8 d1a, by American Type Culture Collection (5 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (5 mentions)
Dmem f12, by Thermo Fisher Scientific (5 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Hcmec d3, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (4 mentions)
L glutamine, by Merck Group (4 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (4 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by American Type Culture Collection (4 mentions)
Bend 3, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by GE Healthcare (3 mentions)
155 protocols using bend 3
1
Immortalized Mouse Brain Endothelial Cell Culture
2
Culturing Immortalized Brain Endothelial Cells
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The immortalized brain endothelial cell line, bEnd.3 (ATCC-CRL-2299), and Dulbecco's modified eagle's medium (DMEM) were obtained from ATCC (USA). The bEnd.3 cells were cultured, according to ATCC-product sheet instruction, in DMEM high glucose medium with L-pyruvate, containing 10 % (v/v) fetal bovine serum and 1% (v/v) of 500 U/ml Penicillin/Streptomycin (Gibco, Germany). Cells were seeded at a density of 5 × 104 cells per cm2 in 24-well plates then incubated at 37°C and 5% CO2 and the culture media was replaced every 3 days.
3
Evaluating Nanoparticle Cytotoxicity and In Vivo Toxicity
4
Coculturing Murine Endothelial Cells and Human Pericytes
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The murine brain endothelial cell line bEnd.3 (ATCC, Manassas, VA, USA) and primary human microvascular pericyte cells (from Dr. Chung‐Hyun Cho, Seoul National University, Seoul, Korea) were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone, Irvine, CA, USA) that was supplemented with 10% fetal bovine serum (FBS; Hyclone), 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin (Sigma‐Aldrich Co.) and incubated at 37 °C in 5% CO2 incubator. For pericyte‐endothelial cell double‐layer culture, Corning Transwell polycarbonate membrane cell culture inserts (6.5‐mm or 24‐mm transwell with 0.4 μm pore; Corning, NY, USA) were used. Pericytes were cultured on the apical layer of transwell (6.5‐mm, 6 × 104 cells cm−2; 24‐mm, 1.8 × 105 cells cm−2), and bEnd.3 cells were seeded on the basolateral layer of transwell (6.5‐mm, 1.2 × 105 cells cm−2; 24‐mm, 3.6 × 105 cells cm−2).
5
Murine Neurovascular Cell Co-Culture
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Complete growth media for all cells (including co-cultures) consisted of DMEM supplemented with 10% FBS and antibiotics. Cryo-preserved bEnd.3 (immortalized murine brain endothelial cells), N2a (immortalized murine brain neuroblastoma), and C8-D1A (immortalized murine astrocytes), were purchased from ATCC. BV-2 (immortalized murine microglia) was obtained from Dr. G. Jean Harry (National Institute of Environmental Health Sciences, Research Triangle Park, NC). bEnd.3, C8-D1A, and BV-2 were maintained as previously described23 (link). N2a cells were differentiated prior to incorporation in co-cultures by replacing complete medium with 0.2% FBS in DMEM for one day after passage. The cells were maintained in medium for 2–3 days followed by replacement with FBS-free DMEM one day prior to co-culture.
6
Culturing bEnd.3 Mouse Brain Endothelial Cells
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The mouse brain endothelial cell line, bEnd.3, was purchased from ATCC (Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (Corning Laboratories, Corning, NY, USA), supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 U/mL streptomycin (Hyclone Laboratories, Logan, UT, USA) at 37°C in a humidified atmosphere mixture of 5% CO2 and 95% air. The medium was replaced every 2 days.
7
Murine Brain Cell Culture Protocol
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The complete culture medium for all cell lines was DMEM supplemented with 10% fetal bovine serum (ATLANTA Biologicals, Flowery Branch, GA, USA) and 1% Penicillin/Streptomycin solution (HyClone Laboratories Inc., South Logan, UT, USA). Murine brain cell lines bEnd.3 (endothelial cells), N2a (neuroblastoma cells), and C8-D1A (astrocytes) were purchased from ATCC. Murine brain cell line BV-2 (microglia) was obtained from Dr. G. Jean Harry (National Institute of Environmental Health Sciences, Research Triangle Park, NC). All cell lines were maintained according to provided protocols and previous studies [5 ]. Briefly, the cells were cultured in 75 cm2 flasks (Corning, NY, USA) separately, and medium was refreshed every other day until the cells reached ≥ 90% confluency.
8
Exosome-mediated Endothelial Cell Function
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The human gastric epithelial cell line GES‐1, human umbilical vein endothelial cells (HUVECs), and mouse brain microvascular endothelial cells (bEND.3; ATCC, Manassas, VA) were cultured in RPMI‐1640, DMEM/F‐12, and DMEM (Gibco/Life Technologies, Grand Island, NY), respectively, supplemented with 10% fetal bovine serum (Biological Industries, Cromwell, CT), 100 U/mL penicillin, and 100 mg/mL streptomycin in a controlled humidified incubator with 5% CO2 as described.35 To evaluate the effect of exosomes on the function of endothelial cells, exosomes (50 μg/mL) from the serum of human subjects or mice with and without CagA+H pylori infection and from conditioned media of GES‐1 cocultured with or without CagA+H pylori for 2 hours were added into the culture system of HUVECs or bEND.3 (see details below for exosome preparation). After culture for 24 hours, HUVECs or bEND.3 were collected for studies on cell migration, tube formation, and proliferation, using a transwell culture system (MCEP24H48; Merck Millipore, Burlington, MA), a Matrigel basement membrane matrix (Cat. No. 356234; Corning Life Sciences, Tewksbury, MA), and a cell‐Light EdU imaging kit (Cat.No.C10310‐1; RiboBio, Guangzhou, China), respectively. GES‐1 was mixed and cultured with H pylori at a multiplicity of infection of 100 for 2, 6, or 12 hours as described.35
9
Endothelial Cell Activation and Inhibition
10
Endothelial Cell Uptake of miR-223 ECVs
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Mouse brain endothelial cells (bEnd.3, ATCC, Manassas, VA) were grown in Dulbecco modified Eagle medium (DMEM, Gibco, Waltham, MA) containing 10% fetal bovine serum (FBS, Gibco, Waltham, MA). To study the transportation of miR-223 from ECVs to endothelial cells, bEnd.3 cells were seeded in 6-well plates. ECVs isolated from WT or miR-223−/y mice as above were added to bEnd.3 cells (500 μl/well). 12 hours after co-culture, the bEnd.3 cells were rinsed 3 times with PBS. Then bEnd.3 cells were collected for further analysis.
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