Sodium alginate (SA), gelatin methacryloyl (GelMA), glycidyl methacrylate (GMA), 2-hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone (Irgacure 2959), sodium carbonate, lithium bromide (LiBr), dialysis cassette (MWCO: 3500), CaCl2, FITC-dextran 20 kDa were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), 100 U mL−1 penicillin/streptomycin, RPMI-1640 medium, 0.25% trypsin-EDTA were purchased from ThermoFisher (USA). Endothelial basal medium (EBM-2) and endothelial growth bullet kit (EGM-2) were purchased from Lonza (Walkersville, MD, USA). Carboxyfluorescein succinimidyl ester (CFSE, ab113853) and ethidium homodimer-1 (ethd-1, E1169) and cell tracker were obtained from Abcam (Cambridge, MA, USA) and ThermoFisher, respectively. 4’,6-diamidino-2-phenylindole (DAPI) was purchased from Life Technologies.
Endothelial basal medium ebm 2
Manufactured by Lonza
Sourced in United States
Endothelial basal medium (EBM-2) is a cell culture medium designed to support the growth and maintenance of endothelial cells. It provides a balanced formulation of nutrients, growth factors, and other essential components required for the optimal in vitro cultivation of endothelial cell types.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Hydrocortisone, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Endothelial growth bullet kit egm 2, by Lonza (2 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Mcf 10a, by American Type Culture Collection (1 mentions)
Atcc htb 22, by American Type Culture Collection (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Gelatin solution, by Merck Group (1 mentions)
Atcc crl 10317, by American Type Culture Collection (1 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Ethylenediaminetetraacetic acid edta, by Merck Group (1 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using endothelial basal medium ebm 2
1
Hydrogel Biomaterials for Cell Culture
2
Isolation and Culture of HUVECs
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The use of umbilical cords for scientific purposes is authorized by the L1211-2 act from the French Public Health Code. Written, informed consent was obtained from each woman who donated an umbilical cord. The privacy of the donor’s personal health information was protected. Human umbilical vein endothelial cells (HUVECs) were isolated according to previously described protocols34 (link). HUVECs were cultured in Endothelial-Basal-Medium (EBM-2, Lonza) supplemented as recommended by the manufacturer. In all experiments, cells were grown until confluence and left for 2 additional days in Labtek 4-chambers (Fisher Scientific). Medium was replaced with fresh medium 16 h before infection, and with fresh non-supplemented medium 1 h before infection.
3
Characterization of Human Cell Lines
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The human brain endothelial cell line (hCMEC/D3) was obtained under licence from University Paris 05, CNRS, Institute Cochin, INSERM (Paris, France). The cell line was maintained and characterised in accordance to regularly updated protocols by Institute Cochin and certified mycoplasma free56 (link). Human breast cancer cell line (MCF-7) (ATCC® HTB-22™) and human mammary epithelial cell line (MCF-10A) (ATCC® CRL-10317™) were purchased from American Type Culture Collection (VA, USA). Endothelial Basal Medium (EBM-2) (#CC-3156) was from Lonza Group Ltd. (Basel, Switzerland). Dulbecco/Vogt Modified Eagle's Minimal Essential Medium (DMEM) (#D0819), basic fibroblast growth factor (#F0291), gelatin solution (#G1393), hydrocortisone (#H-0888), insulin (#I-1882) and phosphate buffered saline (PBS) (#D8537) were purchased from Sigma-Aldrich Co. LLC. (Seelze, Germany). Foetal bovine serum (FBS) (#16000044) and penicillin/streptomycin (#15140122) were obtained from Gibco-BRL, Carlsbad Life Technologies (CA, USA). Recombinant human EGF (AF-100-15) was from PeproTech EC Ltd. (Hamburg, Germany). HEPES (#S11-001) was from PAA Cell Culture Company (Pasching, Austria). The cell culture flasks and plates were purchased from Corning Inc. (NY, USA).
4
Fabrication and Characterization of Bioactive Hydrogels
5
Angiopep-2 Conjugation Protocol
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Dulbecco's Modified Eagle Medium (DMEM)/high glucose was purchased from Gibco corporation, USA. Endothelial Basal Medium (EBM)-2 was from Lonza, USA. Fetal bovine serum (FBS) was obtained from Si Jiqing (Zhejiang, China). Pfu DNA polymerase was purchased from TransGen Biotech (Beijing, China). Various restriction enzymes were purchased from New England Biolabs (Beijing, China). ANG gene was synthesized and pMD18-T-ANG vector was constructed by GenScript Corp (Nanjing, China). T4 DNA ligase and HRP- and FITC-labeled goat anti-human lgG (H + L) secondary antibodies were purchased from Vazyme Biotech (Nanjing, China). Streptococcal protein G agarose beads were purchased from Bestchrom (Shanghai, China). Sulfo-Cyanine 3 maleimide (Cy3) fluorescent dye was purchased from Bioorth Biotech (Nanjing, China). Transwell system was purchased from Corning (NY, USA). Rat-tail collagen type I and FITC-dextran were from Merck KGaA (Darmstadt, Germany). Angiopep-2 gene and primers were synthesized by Genewiz Corporation (Suzhou, China).
6
Endothelial Cell Culture Protocols
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Endothelial basal medium (EBM-2) containing supplements and growth factors was obtained from Lonza (Walkersville, MD, USA). EC medium (ECM) was obtained from ScienCell (San Diego, CA, USA). Fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin and streptomycin were purchased from Gibco (Grand Island, NY, USA). Propranolol was purchased from Sigma (St. Louis, MO, USA). Antibodies against PFKFB3 and β-actin were obtained from Proteintech Biotechnology (Wuhan, China).
7
Immortalized Human Dermal Microvascular Endothelial Cells
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Human dermal microvascular endothelial cells (HMVECs) immortalized with the human telomerase reverse transcriptase catalytic subunit (hTERT) as described by Shao & Guo [40] were graciously received from Dr. Shao. HMVEC hTERT s were grown in Endothelial Basal Medium EBM-2 (Lonza) supplemented with 10% FBS (Gibco), 1 μg ml -1 hydrocortisone (Sigma-Aldrich) and 10 ng ml -1 EGF (Sigma-Aldrich), maintained in a 20% O 2 , 5% CO 2 , 37°C humidified chamber and split regularly 1:4. The absence of mycoplasma infection was confirmed using a detection kit (Lonza). Experiments involving Ang1 were carried out using purified recombinant human Ang1 reconstituted in PBS according to the manufacturer's instructions (R&D Systems).
8
Cultivation of Human Cerebral Microvascular Endothelial Cells
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The Human Cerebral Microvascular Endothelial Cell Line (hCMEC/D3) was obtained from Cellutions Biosystems (CLU512, Ontario, Canada) and maintained at complete EBM-2 medium at 37°C in 5% CO2. Complete medium (final concentration) EBM-2: EBM-2 Endothelial basal medium (Lonza, #190860, Basel, Switzerland), 5% Fetal Bovine Serum (Life Technologies, #14190, Carlsbad, CA); 1% Penicillin-Streptomycin (Life Technologies, #15140–122); 1.4 μM Hydrocortisone (Sigma, #H0135, St. Louis, MO); 5 μg/mL Ascorbic acid (Sigma, #A4544); 1/100 Chemically Defined Lipid Concentrate (Life Technologies, # 11905031); 10 mM HEPES (Life Technologies, #15630–080); 1 ng/mL bFGF (Sigma, #F0291).
Inserts and flasks/Petri dishes were pre-covered with rat Collagen I lower viscosity (R&D Systems, #3443-100-01, Minneapolis, MN). For coating, rat collagen was diluted in 0.02M acetic acid to a final protein concentration of 5 μg/mL. Sufficient amount of solution was added to cover the surface of culture dish and incubate at 37°C for 1 h. This was followed by washing with PBS (Life Technologies, #1653508) three times and then replaced with culture medium. Cells were passed twice weekly and seeded on Petri dishes or flasks at a density of 25,000 cells per cm2. Three-four days after seeding on flasks or Petri dishes, cells reached confluence and can be trypsinized and used until passage 35.
Inserts and flasks/Petri dishes were pre-covered with rat Collagen I lower viscosity (R&D Systems, #3443-100-01, Minneapolis, MN). For coating, rat collagen was diluted in 0.02M acetic acid to a final protein concentration of 5 μg/mL. Sufficient amount of solution was added to cover the surface of culture dish and incubate at 37°C for 1 h. This was followed by washing with PBS (Life Technologies, #1653508) three times and then replaced with culture medium. Cells were passed twice weekly and seeded on Petri dishes or flasks at a density of 25,000 cells per cm2. Three-four days after seeding on flasks or Petri dishes, cells reached confluence and can be trypsinized and used until passage 35.
9
Irradiation of Cerebral Endothelial Cells
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HCMEC/D3 (CELLutions Biosystems Inc), an immortalized cell line from cerebral microvascular endothelial cells, were routinely cultured in EBM-2 endothelial basal medium (Lonza) supplemented with 5% fetal bovine serum, 1% Penicillin/Streptomycin, 10 mM HEPES (Life Technologies), and 1 ng/mL human basic fibroblast growth factor (Sigma-Aldrich) at 37˚C in 5% carbon dioxide and passaged at 90% confluence with trypsin-EDTA. All culture vessels were precoated with 100 µg/mL rat tail collagen (In Vitro Technologies).
For irradiation, cells were seeded at 1x10 4 cells/mL onto collagen-coated 35 mm glass bottom petri dishes (MatTek Corporation) containing 1.5 mL EBM-2 medium and allowed to grow for 2 days to achieve 100% confluency. Cells were treated with a single dose of 5, 15 or 25 Gy of ionizing radiation by linear accelerator (LINAC; Elekta Synergy, Crawley, UK) at Macquarie University Hospital (Sydney, Australia), as previously described [11, 13] . Non-irradiated cells treated identically but without radiation (sham) were used as controls. At 1, 3 or 5 days after radiation or sham treatment, the cells were transferred to the parallel-plate flow chamber.
For irradiation, cells were seeded at 1x10 4 cells/mL onto collagen-coated 35 mm glass bottom petri dishes (MatTek Corporation) containing 1.5 mL EBM-2 medium and allowed to grow for 2 days to achieve 100% confluency. Cells were treated with a single dose of 5, 15 or 25 Gy of ionizing radiation by linear accelerator (LINAC; Elekta Synergy, Crawley, UK) at Macquarie University Hospital (Sydney, Australia), as previously described [11, 13] . Non-irradiated cells treated identically but without radiation (sham) were used as controls. At 1, 3 or 5 days after radiation or sham treatment, the cells were transferred to the parallel-plate flow chamber.
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