C57BL/6 mouse primary brain endothelial cells (mBMEC, #C57-6023) were obtained from Cell Biologics (Illinois, USA). mBMEC (passages no. 4–7, obtained from vendor at P3) were seeded on gelatin coated cell culture flasks or glass chamber slides, cultured in recommended medium (M1168) and maintained at 37 °C with 5% CO2 exposure. Apart from primary cells, an immortalized mouse brain endothelial cell line, bEnd.3 (passage no. 22–25, ATCC® CRL-2299™) obtained from ATCC, VA, USA was also used in in vitro experiments. Immortalized bEnd.3 endothelial cells were seeded on uncoated cell culture flasks as per manufacturer's protocol and maintained at 37 °C with 5% CO2 exposure. Cell culture medium for bEnd.3 consisted of ATCC- formulated Dulbecco's Modified Eagle's Medium ( #30–2002 from ATCC, VA, USA), which was supplemented with 10% FBS (Atlanta Biologicals, GA, USA). The culture medium was changed every other day until the cells reached confluency. Phase contrast microscopy and the expression of characteristic phenotypic markers confirmed the monolayer integrity of both mBMEC and bEnd.3 cells at confluency. On similar notes primary human brain microvascular endothelial cells (HBMEC, obtained from Cell Biologics) were cultured in accordance with the supplier's protocol.
Atcc crl 2299
Manufactured by American Type Culture Collection
Sourced in United States
ATCC® CRL™-2299™ is a cell line derived from human embryonic kidney. It is intended for use in cell culture and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Concanavalin a (cona), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Bend 3, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
Phenol red, by Thermo Fisher Scientific (1 mentions)
S1810 500, by Dutscher (1 mentions)
5 protocols using atcc crl 2299
1
Culturing Mouse and Human Brain Endothelial Cells
2
Culturing bEnd.3 Mouse Brain Endothelial Cells
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The bEnd.3 (Lot.no.: 60323591, ATCC® CRL-2299™) mouse brain microvessel endothelial cell line [34 (link)–36 (link)] was purchased from American Type Culture Collection (Manassas, VA). Cells were incubated in Dulbecco's modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin and maintained at 37°C in a humidified cell incubator with 5% CO2. Cells at passages before 10 were used for the following experiments. Recombinant human HMGB1 was applied to the cell culture medium at different concentrations (10, 100, and 500 ng/mL) for the indicated time.
3
Murine Cell Lines and Splenocyte Isolation
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The transformed C57BL/6 Mus musculus epithelial choroid plexus cell (ECPC-4) cell line (RRID: CVCL_4836) was provided by the RIKEN Bioresource Centre through the National BioResource Project of the AMED, Japan. ECPC-4 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (all from Gibco) at 37 °C/5% CO2.
The murine cerebral brain endothelioma bEnd.3 cell line was obtained from the American Type Tissue Culture Collections (ATCC® CRL™-2299™, Manassas, VA, USA). bEnd.3 cells were cultured in DMEM (ATCC 30-2002) supplemented with 10% FCS, 2 mMl -glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C/5% CO2.
Splenocytes were isolated and seeded at 1 × 106 cells/well in growth medium (DMEM plus 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES, 2 mMl -glutamine, 50 μM 2-mercaptoethanol, and non-essential amino acids; Gibco) in a 96-well plate and incubated for 24 h at 37 °C with 1 µg/mL Concanavalin A (ConA, Sigma-Aldrich) or growth media alone. Treatment with Tet-29 was carried out in both conditions by supplementing the media with 60 µg/mL Tet-29.
The murine cerebral brain endothelioma bEnd.3 cell line was obtained from the American Type Tissue Culture Collections (ATCC® CRL™-2299™, Manassas, VA, USA). bEnd.3 cells were cultured in DMEM (ATCC 30-2002) supplemented with 10% FCS, 2 mM
Splenocytes were isolated and seeded at 1 × 106 cells/well in growth medium (DMEM plus 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES, 2 mM
4
Murine Cell Lines and Splenocyte Isolation
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The transformed C57BL/6 Mus musculus epithelial choroid plexus cell (ECPC-4) cell line (RRID: CVCL_4836) was provided by the RIKEN Bioresource Centre through the National BioResource Project of the AMED, Japan. ECPC-4 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (all from Gibco) at 37 °C/5% CO2.
The murine cerebral brain endothelioma bEnd.3 cell line was obtained from the American Type Tissue Culture Collections (ATCC® CRL™-2299™, Manassas, VA, USA). bEnd.3 cells were cultured in DMEM (ATCC 30-2002) supplemented with 10% FCS, 2 mMl -glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C/5% CO2.
Splenocytes were isolated and seeded at 1 × 106 cells/well in growth medium (DMEM plus 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES, 2 mMl -glutamine, 50 μM 2-mercaptoethanol, and non-essential amino acids; Gibco) in a 96-well plate and incubated for 24 h at 37 °C with 1 µg/mL Concanavalin A (ConA, Sigma-Aldrich) or growth media alone. Treatment with Tet-29 was carried out in both conditions by supplementing the media with 60 µg/mL Tet-29.
The murine cerebral brain endothelioma bEnd.3 cell line was obtained from the American Type Tissue Culture Collections (ATCC® CRL™-2299™, Manassas, VA, USA). bEnd.3 cells were cultured in DMEM (ATCC 30-2002) supplemented with 10% FCS, 2 mM
Splenocytes were isolated and seeded at 1 × 106 cells/well in growth medium (DMEM plus 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES, 2 mM
5
Immortalized Mouse Brain Endothelial Cell Assay
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Immortalized mouse brain endothelial cells, bEnd.3 (ATCC® CRL-2299™, Manassas, Virginia, USA) purchased from Sigma (Sigma-Aldrich, Saint Quentin Fallavier, France) were cultivated in Dulbecco's modified Eagle's medium (DMEM) Glutamax (Gibco, 31966-021), supplemented with 10% foetal bovine serum (FBS, Dutscher S1810-500), 100 U.mL -1 penicillin and 100 μg mL -1 streptomycin (Gibco, 15140122) in a humidified 5% CO2 incubator at 37 °C. Cell line passages < 30 were used for all experiments. To evaluate mortality, metabolic activity and measure ROS, bEnd.3 cells were seeded in 96 well plates at 5×10 4 cells per well (≈ 100 000 cells cm -2 ); for mitochondrial ROS detection, cells were seeded into 24-well plates at a density of 200 000 cells per well (≈ 100 000 cells cm -2 ). Twenty-four hours after, treatments were applied (Glutamate 100 mM, NAC 1 mM, H2O2 2 mM and CNPs at 10, 100, and 1000 µg mL -1 ) diluted in DMEM without FBS and without phenol red (Gibco, 21063-09), and incubated with cells during 4 h or 24 h.
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