The 1321N1 human astrocytoma cell line was cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco), supplemented with 10% foetal bovine serum (FBS) (Sigma) and 1% penicillin/streptomycin (P/S). Human foetal primary astrocytes isolated from the cerebral cortex were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and were cultured in ScienCell medium supplemented with FBS, P/S and Astrocyte Growth Supplement (all ScienCell Research Laboratories). The endothelial cell line, hCMEC/D3 (Cedarlane, USA) was cultured in EBM‐2 medium supplemented with vascular endothelial growth factor, insulin‐like growth factor‐1, 0.025% human epidermal growth factor, 0.1% human basic fibroblast growth factor, 0.04% hydrocortisone, 0.1% ascorbic acid and 2.5% FBS (Lonza) on Collagen‐type 1 (Sigma, UK) coated plates.
Hcmec d3
Manufactured by Cedarlane
Sourced in United States, France, Canada
The HCMEC/D3 is a cell line derived from human cerebral microvascular endothelial cells. It is used as an in vitro model for studying the blood-brain barrier.
Automatically generated - may contain errors
Lab products found in correlation
Endogro mv complete medium, by Merck Group (3 mentions)
Fibroblast growth factor 2 (fgf2), by Merck Group (3 mentions)
Egm 2, by Lonza (2 mentions)
Hcmec d3, by Merck Group (2 mentions)
Astrocyte growth supplement, by ScienCell (1 mentions)
Collagen type 1, by Merck Group (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by ScienCell (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Hdmec, by PromoCell (1 mentions)
Human umbilical vein endothelial cells (huvec), by Corning (1 mentions)
Hcmec d3, by Corning (1 mentions)
T75 culture flask, by Corning (1 mentions)
Huvecs, by ScienCell (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
E coli serotype 0127 b8, by Merck Group (1 mentions)
12 protocols using hcmec d3
1
Culturing Human Astrocytoma and Primary Astrocytes
2
Culturing Human Cerebral Microvascular Endothelial Cells
3
Establishment and Maintenance of Breast Cancer and Endothelial Cell Lines
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MCF-7 and MDA-MB-231 breast cancer cell lines were purchased from ATCC. The brain metastatic MDA-MB-231-BrM2 and the parental cells MDA-MB-231-TGL were obtained from Dr Joan Massagué (Memorial Sloan-Kettering Cancer Center, New York, NY, USA) [17 (link),29 (link)]. Cancer cells were cultured in DMEM medium supplemented with 10% FBS, 1X L-Glutamine and antibiotics (1000 U Penicillin/1000 U Streptomycin). The immortalized human cerebral microvascular endothelial cells (hCMEC/D3) were obtained from Cedarlane (Tebu-Bio, France) and cultured in EndoGRO™-MV Complete Medium (cat# SCME004, EMD Millipore, USA) supplemented with 1ng/mL FGF-2 (cat# GF003, EMD Millipore, USA) and antibiotics. hCMEC/D3 were grown to confluence on tissue flasks precoated with thin collagen I coating (08-115, EMD Millipore) as recommended by the manufacturer. All cells were maintained in a 95% humidified air and 5% CO2 incubator at 37 °C.
4
Culturing Human Brain Endothelial Cells
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The immortalized human CMEC line (hCMEC/D3) (Cedarlane, Burlington, ON, Canada) was chosen as an in vitro model of the BBB as it is widely used and amendable in studying endothelial cell mechanisms with relevance to the brain [72 (link)]. CMECs were inoculated into tissue culture flasks and expanded in endothelial growth medium (EGM-2) (Lonza, Basel, Switzerland). EGM-2 was prepared by supplementing endothelial basal medium (EBM-2) with EGM-2 SingleQuot supplements, which consist of serum, hormones, and growth factors (proliferative and/or angiogenic) that stimulate endothelial cell growth. Medium changes were performed every 2 days, and cells were passaged at 90% confluence.
5
Culturing Immortalized Human Cerebral Microvascular Endothelial Cells
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The immortalized human cerebral microvascular endothelial cell line (hCMEC/D3) was obtained from Cedarlane (Tebu-Bio, France) and maintained in EndoGRO™-MV Complete Medium (cat# SCME004, EMD Millipore, USA) supplemented with 1ng/mL FGF-2 (cat# GF003, EMD Millipore) and antibiotics at 37°C in 5% CO2. Cells were regularly tested for mycoplasma contamination and phenotypic changes.
6
Culturing Endothelial Cell Lines
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HUVECs (ScienCell, USA), hCMEC/D3 (Cedarlane, Canada), hBMEC (Cell Systems, USA), and hDMEC (Promocell, Germany) were cultured in endothelial cell medium (ScienCell, USA) containing 10% fetal bovine serum (FBS), 1% penicillin‐streptomycin (P/S), and 1% endothelial cell growth supplement (ECGS). Frozen stocks were prepared for all cell types after expanding cells in vendor‐recommended media. Frozen stocks of the following passages were thawed and cultured in T75 culture flasks (Corning, USA), where Passage 1 for all cell type indicates the initial stock received from the vendor; HUVEC: Passage 4, hCMEC/D3: Passage 5–7, hBMEC: Passage 6, hDMEC: Passage 3, 2–3 d prior to cell seeding. All cells were cultured in a humidified 37 °C incubator with 5% CO2.
7
Culturing Human Cerebral Microvascular Endothelial Cells
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The human cerebral microvascular endothelial cells (hCMEC/D3) were obtained from Cedarlane (CAN). The hCMEC/D3 cells (passages 28 to 33) were cultured in EBM2 medium supplemented with 5% FBS, hydrocortisone (1.4 µM), ascorbic acid (5 µg/mL), 1% chemically defined lipid concentrate, human bFGF (1 ng/mL), HEPES (10 µM) and antibiotics on collagen I (150 µg/mL)-coated flasks under 5% CO2 at 37 °C. Every 3−4 days, cells were passaged using trypsin/EDTA (Gibco, USA) to detach the cells from the flasks.
8
Cultivation of hCMEC/D3 Endothelial Cells
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The immortalized human CMEC line, hCMEC/D3 (Cedarlane, Burlington, Canada) was grown in endothelial growth medium (EGM-2) (Lonza, Basel, Switzerland). A total of 2 million CMECs were inoculated per T-75 tissue culture flask, and the cells were passaged at 90% confluency (2–3 days of culture) to be used in experiments. Medium was replaced on day 2.
9
Modeling Blood-Brain Barrier with hCMEC/D3 Cells
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Immortalized human cerebral microvascular endothelial cell line (hCMEC/D3) as a model of BBB was purchased from Cedarlane Laboratories, Burlington, Ontario, Canada. The cells were grown in RPMI 1640 medium containing 10% fetal bovine serum (Gibco, 10099141C, Carlsbad, CA), 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO2. For the experiments described hereinafter, hCMEC/D3 cells were exposed to LPS (1 μg/mL, E. coli serotype 0127:B8, Sigma-Aldrich, L4516, St. Louis, Missouri) for 24 h or pretreated with AZD1390 (25 nM), doxorubicin hydrochloride (25 nM), or MK-2206 dihydrochloride (10 μM, MCE, HY-10358, Monmouth Junction, NJ) for 1 h before LPS exposure.
10
Culturing Brain Microvascular Cells
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The human brain microvascular endothelial cell line (hCMEC)/D3 (CLU512, Cedarlane Labs, CA.) was maintained on collagen-coated (50 μg/mL, rat tail type I, BD Biosciences) flasks and grown in VascuLife VEGF – Mv media (LL0005, CellSystems) supplemented with rhVEGF (5 ng/mL), rhEGF (5 ng/mL), rhFGF (5 ng/mL), rhIGF-1 (15 ng/mL), L-Glutamine (10 mM), hydrocortisone hemisuccinate (1 μg/mL), heparin sulphate (0.75 U/mL), ascorbic acid (50 μg/mL), fetal bovine serum (FBS, 5%), 10,000 U/mL penicillin, 10,000 μg/mL streptomycin, and 25 μg/mL amphotericin B. Primary human brain microvascular pericytes and astrocytes (Neuromics, USA), were maintained on poly-L-lysine (10 μg/mL) coated flasks with pericyte (HMP104) and astrocyte (PGB003) growth medium (Neuromics). Astrocytes and pericytes were used at passages 3–5 and hCMEC/D3 at passage 27–29.
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