Some typical human bladder carcinoma cell lines are used for in vitro experiments, such as RT112, RT4, SW780, BIU87, and 5637. We employed an immortalized normal human urothelial cell SV-HUC-1 as normal control to test the biosecurity of harmine. The cells were purchased from the American Type Culture Collection (ATCC, U.S.A.). All cell lines were cultured in DMEM high glucose or F12K medium containing 10% FBS. Cells were cultured in an incubator filled with 5% CO2 at 37 °C. And we used another human normal cell, the primary human umbilical vein endothelial cells (HUVECs) (Science Cell Research Laboratories, San Diego, CA) as a cell model to mimic the process of angiogenesis. HUVECs were cultured in Endothelial Cell Medium (Gibco Life Technology, U.S.A.) supplemented with 5% FBS, 1% endothelial cell growth supplement (ECGS), and 1% penicillin–streptomycin and placed in incubator filled with 5% CO2. Every 2–3 days, the medium for cell culture was refreshed.
Endothelial cell medium (ecm)
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The ECM is a lab equipment product that serves as an electrochemical measurement system. It enables the analysis and characterization of electrochemical processes and materials.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (15 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Huvecs, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Endothelial cell medium (ecm), by ScienCell (4 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Hrmecs, by American Type Culture Collection (2 mentions)
Huvecs, by American Type Culture Collection (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Live dead cell imaging kit, by Thermo Fisher Scientific (1 mentions)
Fv300 microscope, by Olympus (1 mentions)
Penicillin streptomycin antibiotic, by Thermo Fisher Scientific (1 mentions)
Tib 202, by American Type Culture Collection (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Huvecs, by ScienCell (1 mentions)
Ham s f 12k, by American Type Culture Collection (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Hpmecs, by American Type Culture Collection (1 mentions)
30 protocols using endothelial cell medium (ecm)
1
In Vitro Cell Line Cultivation
2
Cell Viability Assessment on Scaffolds
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3T3 fibroblasts (ATCC CRL-1658, Passage-48), HaCat keratinocytes (Passage-54) and EA.hy926 endothelial cells (ATCC CRL-2922, Passage-27) were seeded on pre-sterilized and pre-wetted scaffolds. The membranes were cut into a 1 x 1 cm size and cells were seeded at a final concentration of 2 × 104 cells/sample and cultured in 24-well plates. Both 3T3 fibroblasts and HaCat keratinocytes were maintained in DMEM (Gibco, Ireland) provided with 10% fetal bovine serum and a penicillin/streptomycin solution (Gibco, US origin). EA.hy926 endothelial cells were maintained in endothelial cell medium (Gibco, Ireland). All the cell seeded membranes were incubated in an incubator with a 5% CO2 supply for 24 h, 3 days and 7 days. The LIVE/DEAD Cell Imaging Kit (488/570) was used for the assay according to the protocol described by the manufacturer (Molecular Probes, Invitrogen, USA). After the incubation period, the cells were washed with DPBS and then the prepared reagent (100 µl) was added to each well. After 30 min of incubation at 38°C, the fluorescence images were taken using an Olympus, FV300 microscope.
3
Differentiation and Culture of THP-1 and HUVEC Cells
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The THP-1 cells (ATCC, TIB-202) were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (Gibco), 2-mercaptoethanol of 0.05 mM, and 1% penicillin/streptomycin antibiotic (Gibco), and incubated in a humidified 37°C, 5% CO2 incubator. Phorbol-12-myristate 13-acetate (PMA, 10 nM) (Sigma-Aldrich) was used to differentiate the cells toward a macrophage phenotype after incubation for 72 h. The cells were washed with phosphate-buffered saline (PBS) to remove the PMA-supplemented media and rested in fresh PMA-free media for 24 h before being used for experiments. Human-umbilical vein/vascular endothelium cells (HUVECs) (ATCC, CRL-1730) were cultured in endothelial cell medium (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin antibiotic (Gibco), and incubated in a humidified 37°C, 5% CO2 incubator.
4
Cytotoxicity of Isomangiferin on Breast Cancer Cell Lines
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Human breast carcinoma cell lines used for in vitro experiments included MDA-MB-231, T47D, MCF7, and SKBR3. The 4T1 triple-negative mouse breast cancer cell line was also used. The MCF-10A immortalized normal human mammary epithelial cell line was used as a normal control to test the cancer cell cytotoxicity of isomangiferin. All cells were purchased from the American Type Culture Collection (Manassas, USA) and cultured in high-glucose Dulbecco's modified Eagle's medium or Ham's F12K (Kaighn's) medium containing 10% FBS in an atmosphere of 5% CO2 at 37℃. Primary human umbilical vein endothelial cells (HUVECs; ScienCell Research Laboratories, San Diego, USA) were used as a model to mimic the process of angiogenesis. The HUVECs were cultured in Endothelial Cell Medium (Gibco Life Technology) supplemented with 5% FBS, 1% endothelial cell growth supplement, and 1% penicillin-streptomycin in an atmosphere of 5% CO2. The culture medium was refreshed every 2 to 3 days.
5
Cultivating and Transfecting Human Pulmonary Microvascular Endothelial Cells
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HPMECs were obtained from Shanghai Cell Bank of the Chinese Academy of Science (Shanghai, China) and were cultured in the endothelial cell medium (Gibco, Carlsbad, CA, U.S.A.) containing with 10% heated-inactivated fetal bovine serum (FBS, Gibco), 1% endothelial cell growth factor (Merck, Darmstadt, Germany), 100 IU/ml penicillin (Gibco), and 50 μg/ml streptomycin (Gibco). Then, cells were incubated in a humidified incubator at 37°Cwith 5% CO2.
MiR-218-5p mimic (miR-218-5p), mimic negative control (miR-NC), miR-218-5p inhibitor (in-miR-218-5p) and inhibitor negative control (in-miR-NC) were purchased from RIBOBIO (Guangzhou, China), pcDNA3.1-MIR155HG overexpression vector (MIR155HG), pcDNA3.1-BRD4 overexpression vector (BRD4), pcDNA3.1 empty vector (pcDNA), small interfering RNA (siRNA) against MIR155HG (si-MIR155HG) or siRNA negative control (si-NC) were obtained from Genepharma (Shanghai, China). All the oligonucleotides or vectors were transfected into HPMECs cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. After transfection for 48h, cells were harvested for subsequent analysis.
MiR-218-5p mimic (miR-218-5p), mimic negative control (miR-NC), miR-218-5p inhibitor (in-miR-218-5p) and inhibitor negative control (in-miR-NC) were purchased from RIBOBIO (Guangzhou, China), pcDNA3.1-MIR155HG overexpression vector (MIR155HG), pcDNA3.1-BRD4 overexpression vector (BRD4), pcDNA3.1 empty vector (pcDNA), small interfering RNA (siRNA) against MIR155HG (si-MIR155HG) or siRNA negative control (si-NC) were obtained from Genepharma (Shanghai, China). All the oligonucleotides or vectors were transfected into HPMECs cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. After transfection for 48h, cells were harvested for subsequent analysis.
6
Glucose Effects on hRMEC Culture
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hRMECs were purchased from American Type Culture Collection (Manassas, VA, USA), and were cultured in endothelial cell medium (Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin. The cultures were then maintained in an incubator at 37°C with 5% CO2. hRMECs were treated with 5.5 mM glucose (NG), 5.5 mM glucose, 19.5 mM mannitol (OS), and 25 mM glucose (HG) to examine the effect of glucose on cells.
7
Culturing Human Endothelial Cells
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HPMECs, purchased from ATCC (Manassas, VA, USA), were cultivated in endothelial cell medium (Gibco, USA) containing 10% heated-inactivated fetal bovine serum (FBS, Gibco), 1% endothelial cell growth factor (Beyotime, China), 100 IU/mL penicillin and streptomycin (Sigma) at 37°C in a humidified atmosphere of 5% CO2.
8
Culturing Human Brain Microvascular Endothelial Cells
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Endothelial cell medium (ECM), penicillin, fetal calf serum, trypsin, streptomycin, HBSS, and other culture reagents were purchased from Gibco (Carlsbad, CA, USA). Human brain microvascular endothelial cells (hBMECs) were purchased from ScienCell (Carlsbad, CA, USA). 12-well transwell plates with polystyrene inserts (0.4 μm pore size and 12 mm in diameter) were obtained from Corning Costar (Cambridge, MA, USA). The epithelial voltammeter was obtained from Electrical Resistance System (Millicell ERS-2, Canton, MA, USA). hBMECs cells were maintained in ECM supplemented with 10% fetal bovine serum (FBS),100 units/mL penicillin and 100 mg/mL streptomycin in 5% CO2 at 37 °C.
9
Culturing HUVEC Cells with PolyP Treatments
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HUVEC cells (Lonza, Basel; Switzerland) were cultured in Endothelial Cell Medium (Gibco/Thermo Fisher Scientific, Waltham, MA) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Gibco/Thermo Fisher), 1% penicillin/streptomycin and vascular endothelial growth factor [VEGF] for rapid proliferation, as described [37 (link)]. The cells were cultured at 37°C in humidified 5% CO2, 95% air. The polyP preparations (“Na-polyP[Ca2+]”, “Ca-P particles” or “Ca-polyP-MP”) were added at a final concentration of 30 μg/mL.
After an incubation period for up to 3 d the cells were stained with the far-red fluorescent DNA dye Draq5 (Biostatus, Shepshed; UK) for nuclear visualization or with DAPI (#D9542; Sigma; 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride) for polyP detection [38 ].
After an incubation period for up to 3 d the cells were stained with the far-red fluorescent DNA dye Draq5 (Biostatus, Shepshed; UK) for nuclear visualization or with DAPI (#D9542; Sigma; 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride) for polyP detection [38 ].
10
Stem Cell Characterization and Osteogenic Differentiation
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Human DPSC were obtained from Procell Life Science and Technology Co. (China). To identify stem cell surface markers, flow cytometry detected FITC-conjugated surface antibodies (CD90 (328,107, Biolegend), CD73 (344,015, Biolegend), CD44 (397,517, Biolegend), CD31 (303,103, Biolegend), CD45 (304,005, Biolegend), and CD34 (343,603, Biolegend)). For osteogenic differentiation induction, DPSCs were cultured in a complete medium with 10 mM β-glycerophosphate, 10−7 M dexamethasone, and 50 μg/ml ascorbic acid.
Human umbilical vein endothelial cells (HUVEC) were purchased from Thermo Scientific and cultured in an Endothelial Cell Medium (Gibco). The cell line was qualified post authentication and tested negative for mycoplasma.
Human umbilical vein endothelial cells (HUVEC) were purchased from Thermo Scientific and cultured in an Endothelial Cell Medium (Gibco). The cell line was qualified post authentication and tested negative for mycoplasma.
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