Adult human liver cells were isolated from human liver tissues and cultured as previously described [11 (link),23 (link)]. This study was approved by and followed the guidelines of the Institutional Review Board of Asan Medical Center (IRB number: 2014–1182, Seoul, Korea). Liver tissues were digested using collagenase type I (Worthington Biochemical, Lakewood, NJ, USA). Cells were washed and seeded on fibronectin-coated plates (3 μg/cm2, Sigma Aldrich, St. Louis, MO, USA). Cells were cultured in low-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% antibiotic-antimycotic solution, and Glutamax (Life Technologies, Grand Island, NY, USA) at 37 °C. Upon reaching 85% confluence, cells were split (1:3) using trypsin-EDTA. After propagation, liver cells at passage 5–7 were used for experiments.
Fibronectin coated plate
Manufactured by Merck Group
Sourced in United States, United Kingdom
Fibronectin-coated plates are a type of cell culture substrate designed to promote cell adhesion and growth. Fibronectin is a naturally occurring extracellular matrix protein that facilitates the attachment and spreading of many cell types. These plates provide a standardized and consistent surface for culturing cells in a controlled environment.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (1 mentions)
Collagenase type 1, by Worthington (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
D glucose, by Thermo Fisher Scientific (1 mentions)
Cellbanker 2, by AMS Biotechnology (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Ebm 2 medium, by Cambrex (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Beas 2b, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Beas 2b, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Huvecs, by BD (1 mentions)
Vascular endothelial growth factor vegf, by Thermo Fisher Scientific (1 mentions)
Pd98059, by Merck Group (1 mentions)
Ly294002, by Merck Group (1 mentions)
Endothelial growth media 2, by Lonza (1 mentions)
Smgm 2, by Lonza (1 mentions)
12 protocols using fibronectin coated plate
1
Isolation and Culture of Adult Human Liver Cells
2
Isolation and Transduction of Adult Bone Marrow Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMECs were immunopurified using CD31-captured (Biolegend) magnetic beads (Life Technologies) from digested and lineage depleted (Miltenyi Biotec) adult (12 week) C57BL/6J WBM. BMS were obtained from the CD31− fraction. Resulting BMEC and BMS cultures were transduced with a myristoylated-Akt1 expressing lentivirus (Kobayashi et al., 2010 (link)) and cultured on fibronectin-coated plates (Sigma-Aldrich).
3
Isolation and Expansion of FAA-CPCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To isolate FAA-CPCs, the enzymatically released chondrocytes at a loading concentration of 4000 cells/9.3cm2 were added to fibronectin-coated plates (10μg/ml, Sigma) for 20 min. The removal of non-adherent chondrocytes followed this. The adherent cells were continued in culture for another 12 days till each attached cell achieved five population doublings (each clone > 32 cells). The enriched polyclonal FAA-CPCs were isolated, replated, and expanded in a stromal medium containing 1 ng/ml of recombinant transforming growth factor beta 2 and 5ng/ml human fibroblastic growth factor-2 at 5ng/ml, as per standard established protocol [7 (link)].
4
RNAi Screen Optimization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RNAi screens, Dharmacon (Lafayette, USA) siGENOME SMARTpool oligonucleotide duplexes were used with the exception of Rac1 for which validated SMARTpool oligo14 (link) was used. Results with siGENOME SMARTpools were validated with ON-TARGETplus individual oligonucleotides and shRNAs (Thermo Scientific, Open Biosystems). Sequences of siRNAs and shRNAs are listed in Supplementary Fig. 10 . HUVEC cultured in six-well plates were transfected with 10 nM oligonucleotide duplexes using GeneFECTOR (Venn Nova, Inc.) according to the manufacturer's protocol, and HEK293T cells cultured in six-well plates were transfected with 10 nM oligonucleotide duplexes and 800 ng plasmid DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. HEK293T cells were cultured on fibronectin-coated plates (10 μg ml−1; Sigma-Aldrich) for immunofuorescence and biochemical assays. HUVEC were cultured on fibronectin-coated plates for biochemical assays. Biochemical assays were performed 48 h after transfection.
5
Isolation of Porcine Aortic Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For comparative studies, PAECs were isolated from segments of aorta collected from control pigs (n = 3) terminated following completion of unrelated in vivo studies at TBRC. Upon collection, aortae were placed into DMEM containing 1% P/S and kept at 4 °C until cell isolation (within 24 h). Aortic rings were cut open longitudinally and ECs were collected by lightly scraping the lumen with a scalpel before transfer into EC media (DMEM incl. GlutaMAX-1, 1 g/L D-Glucose, pyruvate (ThermoFisher Scientific, Waltham, MA, USA), 1% P/S, and 10% FBS). ECs were initially cultured in fibronectin-coated plates (100 µg/mL, Sigma-Aldrich, Gillingham, UK), before being passaged into coated T25 flasks by enzymatic detachment (TryplE Express, Gibco, Waltham, MA, USA), then passaged and expanded in non-coated culture flasks until use in experiments, or cryopreserved at −80 °C for later use (CellBanker 2; Amsbio, Abingdon, UK).
6
Isolation and Culture of Rat Mononuclear Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Six to eight week-old male Wistar rats (weight: 220–300 g) were anesthetized via intraperitoneal injection with 1% of sodium phenobarbital (30 mg/kg). Peripheral blood was collected from the heart. Peripheral blood mononuclear cells (MNCs) were isolated by density gradient centrifugation using Ficoll-Paque PLUS (Herause, Germany). The mononuclear cell fraction was carded, washed and centrifuged at 1400 rpm for 10 minutes. The cells were suspended in EBM-2 medium (Clonetics, USA) supplemented with 10% fetal calf serum (FCS, Xiamen Tebao Bioengineering Company), vascular endothelial growth factor (VEGF), human fibroblast growth factor-B (hFGF-B), human epidermal growth factor (hEGF), and plated on rat-derived 10 μg/ml fibronectin-coated plates (Sigma Chemical). After culturing for three days, floating cells were removed by washing with potassium-buffered saline (PBS), and new media was added to the adherent cells. The cells were maintained in culture for seven days2 (link), 3 (link).
7
Culturing BEAS-2B Bronchial Epithelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A commercially available human bronchial epithelial cell line (BEAS-2B) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). BEAS-2B cells were revived in bronchial epithelial growth medium (Cambrex Bio Science, Walkersville, MD, USA) and grown on fibronectin-coated plates (0.5 mg/ mL; Sigma, Gillingham, UK) at 37 ° C in 5% CO 2 and 100% humidity according to the ATCC guidelines. After serial passage culture of the BEAS-2B cells, they were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Life Technologies), L -glutamine (2 m M ), and nonessential amino acid (2 m M ) at 37 ° C in 5% CO 2 and 100% humidity. For tests, cellular suspension of 5 × 10 5 , 1 × 10 5 , and 5 × 10 4 BEAS-2B cells/mL were respectively plated onto 6-, 24-, and 96-well plates and cultured for 24 h before the fiber exposure. Chrysotile provided by Japan Fibrous Materials Research Association was suspended in distilled water by ultrasonic agitation, and processed by autoclave sterilization.
8
Culturing HUVECs and Infecting with Hantavirus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human umbilical vein endothelial cells (HUVECs) were prepared by the method described before [22 (link)]. Cultures of HUVECs were grown on fibronectin-coated plates (Millipore, USA), were maintained in EGM (Lonza, USA) supplemented with 10% fetal bovine serum (PAA, Austria), 100 IU of penicillin/mL, and 100 μg of streptomycin/mL, and were used before the 10th passage.
HTNV strain 76–118 was propagated on Vero E6 cells. Supernatant was collected from cell cultures at 14 days postinfection and was cleared of cell debris by centrifugation at 2,000 ×g and then filtration by 0.45 μm filter. The stocks of HTNV were aliquot and frozen at −80°C. Mock HTNV control was prepared by subjecting HTNV to Co 60 radiation (1 × 104 Gy). For all infections, virus was allowed to adsorb to HUVECs at multiplicity of infection (MOI) of approximately 1 in serum-free EGM maintenance medium for 2 h at 37°C. The cells were then washed and afterwards incubated in EGM growth medium with 10% FBS. The proportion of infected HUVECs was tested by using immunofluorescence. After 48 h postinfection, over 90% of HUVECs expressed viral nucleocapsid protein in the cytoplasm.
HTNV strain 76–118 was propagated on Vero E6 cells. Supernatant was collected from cell cultures at 14 days postinfection and was cleared of cell debris by centrifugation at 2,000 ×g and then filtration by 0.45 μm filter. The stocks of HTNV were aliquot and frozen at −80°C. Mock HTNV control was prepared by subjecting HTNV to Co 60 radiation (1 × 104 Gy). For all infections, virus was allowed to adsorb to HUVECs at multiplicity of infection (MOI) of approximately 1 in serum-free EGM maintenance medium for 2 h at 37°C. The cells were then washed and afterwards incubated in EGM growth medium with 10% FBS. The proportion of infected HUVECs was tested by using immunofluorescence. After 48 h postinfection, over 90% of HUVECs expressed viral nucleocapsid protein in the cytoplasm.
9
Endothelial Differentiation of Umbilical Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
USCs at p3 were plated on fibronectin (Millipore, Billerica, MA) coated 6‐well plates at a density of 3,000 cells/cm2, allowed to attach for 24 hours in the Dulbecco's Modified Eagle Medium(DMEM) with 10% FBS, then cultured in Endothelial Growth Media 2 (EGM‐2; Lonza Biologics, Portsmouth, NH) in 2% FBS with a fresh mix of 50 ng/ml Vascular endothelial growth factor(VEGF) (PeproTech, Rocky Hill, NJ). ECs induced from USCs (EC‐induced USCs) were characterized 14 days after being cultured in EGM‐2 media. As a positive control, HUVECs (BD Bioscience, San Jose, CA) were cultured on fibronectin‐coated plates (Millipore, Billerica, MA) in EGM‐2, while noninduced USCs (p3) in DMEM with 10% FBS were used as a negative control through all the experiments described below. To obtain pure ECs, induced USCs were sorted with fluorescence‐activated cell sorting (FACS) with CD 31 antibody.
To evaluate the roles of the classic regulation pathways13 (phosphatidylinositol 3‐kinase (PI3K) and mitogen‐activated protein kinase(MAPK) pathways) of endothelial differentiation when USCs induced into ECs, the PI3K inhibitor LY294002 (Sigma, St. Louis, MO) and MAPK inhibitor PD98059 (Sigma, St. Louis, MO) were added to the EGM‐2, each at a final concentration of 15 µM.
To evaluate the roles of the classic regulation pathways
10
Isolation and Culture of Valvular Interstitial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pathological VICs (VIC p ) and control VICs (VIC c ) were obtained after type-I collagenase digestion of the aortic valve, as previously described, 15 (link) from patients after surgery and after autopsy for controls, respectively. VICs were seeded on fibronectin-coated plates (0.4 μg/cm 2 , Millipore, Billerica, MA), cultured in SMGM-2 (smooth muscle growth medium-2; Lonza, Walkersville, MD) supplemented with 20% fetal bovine serum (Hyclone, Logan, UT) and used between passages 2 to 6. The absence of contamination by ECs and macrophages was ensured after digestion by flow cytometry with fluorochrome-conjugated antibodies CD31 (BD Biosciences, San Jose, CA) and CD68 (BD Biosciences). All assays were performed at 37 °C, on 95% sterile air, and 5% CO 2 in a saturated humidified incubator.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!