To confirm the relationship between EMPs and the P38 MAPK pathway, we used targeted siRNAs to reduce expression of P38 MAPK in HUVECs. HUVECs were cultured in endothelial cell medium (ScienCell) supplemented with 5% Fetal bovine serum (FBS), 1% growth factors, and 1% penicillin/streptomycin. Cells were serum starved in 0.5% FBS over night before experiments. Targeted SiRNAs and scrambled siRNAs (negative control) (Dharmacon, MA) were transfected into cells with siPORT™ NeoFX™ Transfection Agent (Invitrogen, USA) and Opti-MEM I (Gibco). InitialsiRNA concentrations used were 70 nM. At 12 h after transfection, medium was exchanged with endothelial cell medium (Sciencell, Carlsbad, CA) consisting of 10% Fetal bovine serum and 10 ng/mL epidermal growth factor to remove siRNA. A separate control group containing only with siPORT™ NeoFX™ Transfection Agent and Opti-MEM I was also employed.
Endothelial cell medium (ecm)
Manufactured by ScienCell
Sourced in United States, China, Germany, Japan, Canada
ECM is a laboratory product that provides an extracellular matrix for cell culture applications. It serves as a substrate to support the growth and attachment of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (143 mentions)
Huvecs, by ScienCell (118 mentions)
Endothelial cell growth supplement (ecgs), by ScienCell (91 mentions)
Fetal bovine serum (fbs), by ScienCell (79 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (56 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (34 mentions)
Penicillin streptomycin, by ScienCell (26 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (26 mentions)
Huvecs, by American Type Culture Collection (23 mentions)
Penicillin, by Thermo Fisher Scientific (22 mentions)
Rpmi 1640, by Thermo Fisher Scientific (21 mentions)
Streptomycin, by Thermo Fisher Scientific (20 mentions)
Streptomycin, by ScienCell (17 mentions)
Penicillin, by ScienCell (16 mentions)
Human umbilical vein endothelial cells (huvec), by ScienCell (15 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (14 mentions)
Hbmec, by ScienCell (13 mentions)
Trypsin edta, by Thermo Fisher Scientific (12 mentions)
Matrigel, by BD (11 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (10 mentions)
724 protocols using endothelial cell medium (ecm)
1
Investigating the Role of P38 MAPK in EMP Regulation
2
Culturing HUVECs and HASMCs
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HUVECs and HASMCs were cultured in 25 cm2 cell culture flasks at 37 °C in a humidified atmosphere with 5% CO2 and specialized mediums (endothelial cell medium, ECM, smooth muscle cell medium, SMCM) were purchased from ScienCell Research Laboratories, Inc. (San Diego, CA, USA).
3
Endothelial Cell Culture and rLECT2 Treatment
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HRMECs were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology (#ZQ0884; Shanghai, China) and cultured in endothelial cell medium (ECM, #1001; ScienCell Research Laboratories, Carlsbad, CA, USA) containing 5% fetal bovine serum (FBS), 100 units of penicillin, and 100 µg streptomycin per milliliter of medium. HUVECs were acquired from ScienCell Research Laboratories (#8000) and routinely cultured in ECM containing 5% FBS, 100 units of penicillin, and 100 µg streptomycin per milliliter of medium. Both HRMECs and HUVECs (passages 3–5) were grown on attachment factor (mouse tail collagen)–coated tissue-culture-grade plasticware and maintained in a humidified atmosphere of 5% CO2/95% air at 37°C. The confluent cells were treated with or without 30-mM glucose (high glucose to simulate hyperglycemia) for up to 72 hours, and mannitol (30 mmol/L) was used as a high-osmolarity control. In the experiment, the cells were treated with rLECT2 (40 ng/mL, 80 ng/mL, 160 ng/mL, or 320 ng/mL). The subsequent experiments are described below. The protein samples were stored at −80°C until use.
4
HUVEC Preconditioning and Culture
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Human umbilical vein endothelial cells (HUVECs; ScienCell, USA) were cultured in endothelial cell medium (ECM; ScienCell, USA) with 5% fetal bovine serum (FBS; ScienCell, USA), 1% endothelial cell growth supplement (ECGS; ScienCell, USA), and 1% penicillin/streptomycin solution (P/S; ScienCell, USA) in 37 °C incubator with 5% CO2 and 95% air. HUVECs were passaged when the cells reached 80–90% confluency. Cells were used for subsequent experiments at passages 4–7. The HUVECs were preconditioned by LV-FGF-2+-hGMSC-CM, hGMSC-CM, or LV-vector+-hGMSC-CM for 3 days, and ECM (ScienCell, USA) without CM was chosen as the negative control. Then, the cells were cultured by ECM with 5% fetal bovine serum (FBS; ScienCell, USA), 1% endothelial cell growth supplement (ECGS; ScienCell, USA), and 1% penicillin/streptomycin solution (P/S, ScienCell) for 7 to 10 days.
5
Regulation of HRGEC by miR-34a and Autophagy
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Human renal glomerular endothelial cells (HRGEC, USA) were purchased from ScienCell Research Laboratories Company. Cells were cultured with endothelial cell medium (ECM, ScienCell, USA) supplemented with 10% fetal bovine serum for 2-3 days and cultured with serum-free ECM when cells were fused up to 80% for 24 hours for standby use. Then, the cells received the following treatment: normal glucose (5.6 mmol/L glucose), high concentration glucose (10, 15, 20, and 30 mmol/L), miR-34a mimic, miR-34a inhibitor, rapamycin (autophagy inducer), and DC661 (autophagy inhibitor).
According to the manufacturer's protocol, miR-34a mimic (50 nmol) and miR-34a inhibitor (100 nmol, RiboBio, China) were transiently transfected into HRGEC using riboFECT™ CP transfection agent and collected after culture for 48 hours. Incubation with the transfection reagent alone served as a control.
According to the manufacturer's protocol, miR-34a mimic (50 nmol) and miR-34a inhibitor (100 nmol, RiboBio, China) were transiently transfected into HRGEC using riboFECT™ CP transfection agent and collected after culture for 48 hours. Incubation with the transfection reagent alone served as a control.
6
Culturing Human Endothelial Cells
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Human retinal endothelial cells (HRECs) and Human umbilical vein endothelial cells (HUVECs) were purchased from ScienceCell (Carlsbad, CA) and maintained in ECM according to company’s instructions. Specifically, ECM (ScienCell) supplemented with 5% FBS and endothelial cell growth supplement (ECGS, ScienCell) according to company’s instructions.
7
Isolation and Culture of HUVECs
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Human umbilical vein endothelial cells (HUVECs) were harvested from fresh umbilical cords following a previously established protocol [14 (link)]. Fresh umbilical veins were digested with 0.1% (w/v) collagenase type 1A solution at 37°C for 15 min to release endothelial cells from the vessel walls. And then, the vessels incubated with collagenase type 1A were rinsed with ECM (ScienCell, San Diego, USA); HUVECs were collected by centrifugation at 1000 rpm for 5 min and cultured in ECM. HUVECs were used at passage 3.
8
NSCLC Cell Line Cultivation and Hypoxia Exposure
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The NSCLC cell line NCI-H520 was purchased from the American Type Culture Collection (Manassas, VA, USA). The A549, NCI-H460, and NCI-H1975 cell lines were purchased from the Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The NSCLC cell lines were maintained in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% fetal bovine serum (FBS) and 5 mg/mL penicillin/streptomycin at 37 °C. Human pulmonary microvascular endothelial cells (HPMEC) were purchased from ScienCell Research Laboratories (San Diego, CA, USA) and maintained in endothelial cell medium (ScienCell) supplemented with 10% FBS and 5 mg/mL penicillin/streptomycin at 37 °C. Cells under normoxic conditions were incubated at 21% O2 and 5% CO2. Cells under hypoxic conditions were incubated at 1% O2 and 5% CO2.
9
Endothelial Cell Inflammatory Response to IgG Antibodies
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HRGEC were first seeded on BPF-coated 24-well plates at a concentration of 5 × 104 cells/well. When the cellular growth became confluent, the supernatants were removed. Each well was then washed by PBS and incubated with serum-free Endothelial Cell Medium (ScienCell Research Laboratories, CA, USA). Patient-derived IgG monoclonal antibodies and their corresponding IgG isotype controls at different concentrations (final conc. 100 μg/ml, 50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, 0 μg/ml) were individually added to each well at 37 °C. Twenty-four hours later, the supernatants were collected for the analysis of interleukin (IL)-1, IL-6, IL-8, monocyte chemoattractant protein (MCP)-1, interferon (IFN)-γ, and IFN-α (IL-1, 6, 8; MCP-1; and IFN-γ detected by DuoSet ELISA Kits, R&D Systems, Inc., Minneapolis, USA; IFN-α detected by Matched Antibody Pair Kit, Eugene, OR, USA). Moreover, in the experiment of endothelial IFN-α production, some HRGEC were pre-treated with DNAse I. The effects of selected dsDNA-reactive monoclonal anti-HRGEC antibodies on IFN-α production by DNAse I-treated HRGEC were evaluated.
10
Isolation and Culture of HUVECs
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Human umbilical vein cells (HUVECs) were isolated by collagenase treatment of vessels as previously described,17 (link) and cultured in endothelial cell medium (ScienCell, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum, 1% endothelial cell growth supplement (ScienCell, Carlsbad, CA, USA) and penicillin (100 U/mL) + streptomycin (100 μg/mL). HUVECs were used in 2th- 6th passage. Each subject provided signed written informed consent and the study was approved by the ethics committee of the First Affiliated Hospital of Nanchang University.
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