Recombinant human omentin was purchased from Biovendor (Candler, NC, USA). EndoGRO Basal Medium supplemented with kit, H2O2 and 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) were purchased from Merck Millipore (Darmstadt, Germany). 3-(4,5-dimethylthiazol-2-yl) -2,5-dephenyltetrazolium bromide (MTT) and Hoechst 33358 were purchased from Sigma Co. (St. Louis, MO, USA). 2.5% Trypsin-EDTA solution 10× was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Annexin V/PI assay kit and GPx assay kits were purchased from Trevigen (Gaithersburg, MD, USA). All the other chemicals used were of analytical grade.
Endogro basal medium
Manufactured by Merck Group
Sourced in Germany
EndoGRO basal medium is a cell culture media formulation designed to support the growth and maintenance of endothelial cells. It provides the necessary nutrients and components to facilitate the in vitro culture of these specialized cell types.
Automatically generated - may contain errors
Lab products found in correlation
Oligofectamine, by Thermo Fisher Scientific (2 mentions)
2 7 dichlorodihydrofluorescein diacetate h2dcf da, by Merck Group (1 mentions)
Hoechst 33358, by Merck Group (1 mentions)
Human tnf α, by R&D Systems (1 mentions)
Polyfect, by Qiagen (1 mentions)
Huvecs, by Thermo Fisher Scientific (1 mentions)
Huvecs, by Lonza (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Biowest (1 mentions)
Matrigel, by BD (1 mentions)
6 protocols using endogro basal medium
1
Evaluating Omentin's Antioxidant Effects
2
Culturing Immortalized Human Brain Endothelial Cells
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Collagen gels were prepared at a collagen concentration of 150 μg/ml collagen type I, rat tail (Merck Millipore, Darmstadt, Germany) in PBS. To allow collagen gel formation, the flask was incubated at 37°C for 1 h in a humidified atmosphere.
Immortalized human cerebral microvascular endothelial hCMEC/D3 cells were maintained in 75 cm3 flasks precoated with collagen in EndoGRO Basal Medium (Merck Millipore, Darmstadt, Germany) supplemented with 1 ng/ml human fibroblast growth factor basic protein (FGF-2) (Merck Millipore, Darmstadt, Germany), 0.2% EndoGRO-LS supplement, 5 ng/ml recombinant human epidermal growth factor, 50 μg/ml ascorbic acid, 10 mMl -glutamine, 1 μg/ml hydrocortisone hemisuccinate, 0.75 U/mL heparin sulfate, 2% (v/v) fetal bovine serum (FBS), 100 units/mL of penicillin and 100 μg/ml streptomycin. The cells are cultivated at 37°C in a humidified atmosphere with 5% CO2.
Immortalized human cerebral microvascular endothelial hCMEC/D3 cells were maintained in 75 cm3 flasks precoated with collagen in EndoGRO Basal Medium (Merck Millipore, Darmstadt, Germany) supplemented with 1 ng/ml human fibroblast growth factor basic protein (FGF-2) (Merck Millipore, Darmstadt, Germany), 0.2% EndoGRO-LS supplement, 5 ng/ml recombinant human epidermal growth factor, 50 μg/ml ascorbic acid, 10 mM
3
Culturing and Transfecting Endothelial and Kidney Cells
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Human umbilical vein endothelial cells (HUVECs) were grown on 0.1% gelatin coating in EndoGRO basal medium (Millipore) containing 5% fetal bovine serum (FBS) as previously described30 (link). Passage number did not exceed P7. Human epithelial kidney cells (HEK293) were used in promoter activity experiments. HEK293 were grown in 10% FBS DMEM media. Human TNFα (R&D Systems) was used in all in vitro experiments at 10ng/ml. Protein knockdown by siRNA (Ambion) in HUVECs was done by Oligofectamine (Invitrogen) for 3h and followed by 48h growth. Transfection of HEK293 by plasmid DNA was accomplished through Polyfect (Qiagen) according to manufacturer protocol. Transfection was allowed for 18h prior to treatment with harvest or TNFα treatment. THP-1 monocytes were grown in RPMI media containing 10% FBS.
4
Endothelial Cell Culture and Inflammation Assay
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Human umbilical vein endothelial cells (HUVECs) (Lonza, Portsmouth, NH, USA) were cultured in a flask coated with 0.1% gelatin using EndoGRO basal medium (Millipore, Burlington, MA, USA) containing 5% fetal bovine serum (FBS) and used for experiments until Passage 6. Human aortic endothelial cells (HAECs) (Cell Application Inc. San Diego, CA, USA) were grown in human endothelial cell growth medium (Cell Application Inc.) containing 10% FBS and used for experiments until Passage 7. Proinflammatory cytokine cocktail containing TNF-α (10 ng/mL), IL-1β (10 ng/mL), and IL-6 (10 ng/mL) was used in in vitro experiments. siRNAs were transfected using Oligofectamine (Invitrogen, Waltham, MA, USA) in HUVECs for 3 h, followed by 48 h growth. The sequence for human CD137 siRNA is 5′-AAGCAGTTACTACAAGGATCC-3′; human CSF1 siRNA is 5′-GAUCCAGUGUGCUACCUUAAGAAGG-3′; human IL5RA siRNA is 5′-GCAAAGGAAUGUUAAUCUAGAAUAT-3′. Human Atox1 siRNA was purchased from Ambion (Austin, TX, USA). THP-1 monocyte cells were grown in RPMI medium with 10% FBS.
5
Recreating Blood Vessel-Tumor Interaction
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Human umbilical
vein endothelial
cells (Promocell) were used to recreate blood vessel lining in the
blood vessel model channel, and HeLa cells were used in to create
tumor spheroids. HUVECs were cultured in EndoGRO Basal medium (Millipore)
supplemented with SCME001 kit (EndoGRO-LS Supplement 0.2%, rh EGF
5 ng/mL, ascorbic acid 50 μg/mL,l -glutamine 10 mM,
hydrocortisone hemisuccinate 1 μg/mL, heparin sulfate 0.75 U/mL,
FBS 2%), and penicillin/streptomycin 1% (Biowest). HeLa cells were
cultured in Dulbecco’s modified Eagle medium (DMEM, as received
withl -glutamine, 4.5 g/L d -glucose and pyruvate,
Gibco) supplemented with FBS 5% (Gibco) and penicillin/streptomycin
1% (Biowest). HUVECs were cultured in 75 cm2 flasks and
HeLa in 25 cm2 flasks at 37 °C and 5% CO2. Cells were harvested using trypsin-EDTA (0.25%, Gibco) when they
reached 70–80% confluence.
vein endothelial
cells (Promocell) were used to recreate blood vessel lining in the
blood vessel model channel, and HeLa cells were used in to create
tumor spheroids. HUVECs were cultured in EndoGRO Basal medium (Millipore)
supplemented with SCME001 kit (EndoGRO-LS Supplement 0.2%, rh EGF
5 ng/mL, ascorbic acid 50 μg/mL,
hydrocortisone hemisuccinate 1 μg/mL, heparin sulfate 0.75 U/mL,
FBS 2%), and penicillin/streptomycin 1% (Biowest). HeLa cells were
cultured in Dulbecco’s modified Eagle medium (DMEM, as received
with
Gibco) supplemented with FBS 5% (Gibco) and penicillin/streptomycin
1% (Biowest). HUVECs were cultured in 75 cm2 flasks and
HeLa in 25 cm2 flasks at 37 °C and 5% CO2. Cells were harvested using trypsin-EDTA (0.25%, Gibco) when they
reached 70–80% confluence.
6
HUVEC Cell Culture for Vascular Research
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HUVEC cells are primary cells extracted from human neonatal umbilical cords used for many vascular biology research applications, such as inflammation, angiogenesis and blood clotting. The cells were purchased from Millipore™. The culture was maintained in a sub-confluent state in culture Petri Dish (113 2 ) coated with fibronectin at 37 • C. HUVEC cells were grown in EndoGro basal medium (Millipore™) supplemented with 5 ng/mL of rhVEGF, rhEGF, rhFGFb respectively, 15 ng/mL rh IGF-1, 10 mM L-glutamine, 0.75 U/mL heparin sulfate, 1 µg/mL hyroscortisone hemisucinate, 50 µg/mL ascorbic acid, and 10% FBS.
For the final cell culture, the cells were grown in Matrigel ® (BD Biosciences) according to a no top-coat protocol. For polymerisation, Matrigel ® was incubated for 30 minutes at 37 • C and 4000 to 10000 cells were seeded and allowed to adhere for approximately 45 minutes. Then their complete medium was slowly poured over the attached cells.
All cells were routinely cultured in a humidified atmosphere with 5% CO 2 at 37 • C for a day.
For the final cell culture, the cells were grown in Matrigel ® (BD Biosciences) according to a no top-coat protocol. For polymerisation, Matrigel ® was incubated for 30 minutes at 37 • C and 4000 to 10000 cells were seeded and allowed to adhere for approximately 45 minutes. Then their complete medium was slowly poured over the attached cells.
All cells were routinely cultured in a humidified atmosphere with 5% CO 2 at 37 • C for a day.
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