Endothelial cell monolayer disruption (transmigration) by monocytes +/− LPS and TLR2 ligand stimulation was measured using the 9600Z Electrical Cell-Substrate Impedance Sensing (ECIS) system (Applied BioPhysics, Troy, NY, USA). A 96-well plate containing 20 gold film electrodes per well (ibidi) was coated with 1 mg/mL neutralized rat tail collagen type I (Thermo Fisher Scientific) for 10 min at room temperature and 2 µg/mL bovine plasma fibronectin (Thermo Fisher Scientific) for 45 min at 37 °C and 5% CO2. Wells were then inoculated with LECs at a seeding density of 30,000 cells per well in 100 µL huMEC complete media at 37 °C and 5% CO2. The run was performed under a single frequency of 4000 Hz and continuously monitored every 60 s. Impedance (Z) was determined by Ohm’s law: Z = V/I. After 20 h, when an endothelial monolayer was reached, KO and WT THP-1 cells with and without stimulants were added at a density of 100,000 cells per well in 50 µL huMEC complete media. For stimulation, 100 ng/mL LPS (Thermo Fisher Scientific) or 300 ng/mL of either TLR2-specific ligand Pam2CSK4 (Pam2) or Pam3CSK4 (Pam3) (both InvivoGen) was used. Endothelial monolayer disruption was observed for 10 h.
Bovine plasma fibronectin
Manufactured by Thermo Fisher Scientific
Sourced in Spain, United States
Bovine plasma fibronectin is a glycoprotein derived from bovine plasma. It is a key component of the extracellular matrix and plays a role in cell adhesion, migration, and wound healing.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Neutralized rat tail collagen type 1, by Thermo Fisher Scientific (2 mentions)
Pam3csk4 pam3, by InvivoGen (1 mentions)
Pam2csk4 pam2, by InvivoGen (1 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (1 mentions)
Recombinant human sdc4, by R&D Systems (1 mentions)
Recombinant human decorin, by R&D Systems (1 mentions)
3 aminopropyltriethoxysilane aptes, by Merck Group (1 mentions)
Sulfuric acid, by Merck Group (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
96w20idf pet, by Ibidi (1 mentions)
Pluronic acid f 127, by Merck Group (1 mentions)
Mem non essential amino acid, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Arpe 19, by American Type Culture Collection (1 mentions)
Matrigel, by Zeiss (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Lsm 510 confocal laser scanning microscope, by Zeiss (1 mentions)
13 protocols using bovine plasma fibronectin
1
Endothelial Barrier Disruption by Monocyte Transmigration
2
Functionalization of Substrates and Probes
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Recombinant human decorin, recombinant human α5β1-integrin, and recombinant human SDC4 were purchased from R&D Systems (Minneapolis, MN). Bovine plasma fibronectin was purchased from Thermo Fisher Scientific (Waltham, MD). HS was purchased from Iduron (Manchester, UK). Hydrogen peroxide, sulfuric acid, (3-aminopropyl)triethoxysilane (APTES), and N-hydroxysuccinimide-poly(ethylene glycol)-maleimide (NHS-PEG-Mal) were purchased from Sigma-Aldrich (St Louis, MO).
Before substrates or probes were functionalized, they were cleaned using isopropanol, followed by 5 min in an oxygen plasma, and finally with deionized water.
Before substrates or probes were functionalized, they were cleaned using isopropanol, followed by 5 min in an oxygen plasma, and finally with deionized water.
3
Assessing Endothelial Cell Barrier Integrity
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An EC monolayer breakdown (transmigration) assay was performed using the electrical cell-substrate impedance sensing (ECIS) model 9600Z (Applied BioPhysics, Troy, NY, USA). Opto-TLR4-LOV LECs, opto-TLR4 ΔECD2-LOV LECs and opto-TLR4-LOV HUVECs were seeded at a density of 40,000 cells/100 µL of complete medium onto a 96-well plate containing 20 gold film electrodes per well (96W20idf PET; ibidi, Gräfelfing, Germany; 72098) pre-coated with 1 mg/mL neutralized rat tail collagen type I (Thermo Fisher Scientific, Vienna, Austria; A1048301) at room temperature for 10 min, and 2 µg/mL bovine plasma fibronectin (Thermo Fisher Scientific, Vienna, Austria; 33010018) at 37 °C for 45 min. A small-amplitude AC signal (4000 Hz) (I) was imposed across the electrodes, onto which cells were attached, resulting in a potential (V) across the electrodes that was measured using the ECIS instrument [24 (link)]. The impedance (Z) was determined by Ohm’s law Z=V/I. ECs were grown to confluent monolayers for 27 h before being treated with blue light, LPS (100 ng/mL), 50,000 c/well THP-1, 50,000 c/well THP-1 M0, 50,000 c/well PBMC, combinations of blue light or LPS, and the mentioned cell types, or left untreated. EC breakdown was assessed by continuous resistance measurement for 3 h.
4
MDCK Epithelial Colony Growth on PDMS
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The parental MDCK cell line (canine epithelial kidney) was obtained from the Cell Bank of the State of Rio de Janeiro (BCRJ, cell code 0168). MDCK were cultured in Dulbecco’s Modified Eagle Medium (Sigma D6046) low glucose, supplemented with 10% Fetal Bovine Serum (Gibco, lot #210415K), MEM Non-essential Amino Acid (Sigma, M7145), L-glutamine 2 mM (Sigma, G7513). For the growth of epithelial colonies over PDMS substrates, PDMS substrates are sterilized with ethanol and coated with bovine plasma fibronectin (ThermoFischer Scientific, 33010018 20 μg mL−1) for 1 h at room temperature. In parallel, small PDMS blocks with a central, custom-made well are first sterilized and passivated with a 5% solution of BSA in water and then a 5% solution of Pluronic acid F-127 (Sigma, P2443) and placed over the PDMS substrate. A MDCK suspension (1.15 million cells mL−1) is loaded into the wells incubated overnight before carefully removing the wells.
5
ARPE-19 Cell Differentiation Protocol
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The human retinal pigment epithelial cell line (ARPE-19) obtained from ATCC (Manassas, VA, USA) was seeded on the upper side of Costar transwells membrane inserts with 8 µm pores (Corning, St. Louis, MO, USA) which were pre-coated previously with bovine plasma fibronectin (Invitrogen, Thermofisher Scientific, Waltham, MA, USA). ARPE-19 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics in a humidified atmosphere of 5% CO2 at 37 °C for 7–8 weeks. Passage 23–27 cells were used for the experiments.
6
Chick Embryo Cranial Neural Crest Isolation
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Studies involving these early stages of chick embryos do not require review by the UNC Animal Care and Use Committee. Fertile chicken eggs (strain Hampshire Red, Department of Poultry Science, North Carolina State University, Raleigh NC) were incubated to the 4-8 somite stage. The early crania were dissected free and incubated in F12 containing 10% fetal calf serum (heat-inactivated) and 7.5% Chick Embryo Extract (prepared from 10-day-old embryos as per) [38, 39] , and 1× penicillin-streptomycin, and containing alcohol concentrations from 0 to 80 mM. After 2 hr, isolated crania were gently washed several times in alcohol-free media and immediately explanted as below.
Primary CNCs were prepared from the above crania using explant cultures as described [32] . In brief, isolated crania from above were placed dorsal side down atop glass coverslips that had been pretreated with bovine plasma fibronectin (25 ug/ml, Invitrogen), then incubated (37°C under 5% CO2) in F12 medium containing 10% heat-inactivated fetal bovine serum, 1× penicillin-streptomycin, and 7.5% chick embryo extract. After 18 hr incubation, cranial tissue was gently lifted away using fine forceps and migrated cells were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for subsequent immunostaining and cell quantitation.
Primary CNCs were prepared from the above crania using explant cultures as described [32] . In brief, isolated crania from above were placed dorsal side down atop glass coverslips that had been pretreated with bovine plasma fibronectin (25 ug/ml, Invitrogen), then incubated (37°C under 5% CO2) in F12 medium containing 10% heat-inactivated fetal bovine serum, 1× penicillin-streptomycin, and 7.5% chick embryo extract. After 18 hr incubation, cranial tissue was gently lifted away using fine forceps and migrated cells were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for subsequent immunostaining and cell quantitation.
7
Investigating Macrophage Invasion under Hypoxia
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The 3D inverted invasion assay was performed as previously described35 (link). Briefly, 100 mL of Matrigel (BD Biosciences), mixed 1:1 with ice-cold PBSand supplemented with 50 mg/mL bovine plasma fibronectin (Invitrogen), was transferred to a transwellinsert (8-mm pore; Corning Incorporated, Corning, NY, USA) to polymerise at 37 °C. After polymerisation, transwell inserts were inverted, and 5 × 104 WT or HIF-1α KO BMDMs were applied directly to the lower side of the filter insert and allowed to adhere for 2 hours. Finally, inserts were placed into a chamber containing macrophage medium and incubated for 72 hours at 37 °C and 5% CO2 under either normoxic (20.9% O2) or hypoxic (1% O2) conditions for invasion into the gel. Cells were then stained with 4 mM calcein-AM (Invitrogen). Serial confocal optical sections of the Matrigel/fibronectin gel were captured with a Zeiss LSM510 laser scanning confocal microscope (Zeiss). For each experimental condition, the inverted invasion assay was performed in duplicate, and confocal data were collected from 3 fields of view per transwell (total, 6 fields of view). Experiments were repeated with cells independently isolated from 3 mice per genotype. Invasion data were quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
8
Fibroblast seeding on hydrogel constructs
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Normal human dermal fibroblasts, nhDF (Lonza, CC‐2511), were grown in Dulbecco's Medium Eagle's Medium (DMEM 41966‐029, Thermofisher) supplemented with 10% fetal bovine serum (FBS; 758093, Serana) and 1% penicillin–streptomycin (15140163, Gibco). Cells were cultured in T‐25 cell flasks at 37°C in a humidified atmosphere with 5% CO2. Prior to cell seeding, hydrogel constructs were sterilized by 20 min UV exposure (λ = 254 nm) and coated with 10 µg mL−1 fibronectin in PBS by a 30 min incubation at room temperature (bovine plasma fibronectin, 1 mg mL−1, 33010–018, Invitrogen). Fibroblasts were dissociated using 0.05% trypsin/Ethylenediaminetetraacetic acid (EDTA) with phenol red (25300054, Gibco). A total of 22000 cells were seeded on each construct. After overnight adhesion, topographical patterns were induced as described above. All experiments were performed with cells until passage 15.
9
Adsorption of Bovine Proteins on Silicon Oxide
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Bovine serum albumin, HPLC grade water (resistivity > 18 MΩ cm), phosphate buffered saline (PBS) and sodium deodecyl sulphate (SDS) were purchased from Sigma (Sigma & Aldrich) and used as received. Bovine plasma Fibronectin was purchased from Life Technologies™. Parylene-C was purchased as a dimer from SCS™. Hellmanex® III (Hellma Analytics) (2% in deionized water) was used to clean the DPI injection loops. Untreated silicon oxynitride (SiON) multiple path length dual polarization interferometer (MPL-DPI) chips (Farfield-Biolin Scientific AB) are composed of approximately 11% of silicon nitride and 89% of silicon dioxide (SiO2). Comparison of surface coverage values of Bovine serum albumin adsorbed onto SiON and SiO2 show no significant difference (Table S1) [26] . Silicon oxynitride DPI sensor surface and silicon dioxide surfaces are therefore referred as silicon oxide surfaces in the manuscript. Details of cleaning procedures can be found in our previous work [22] (link). As previously reported, contact angle measurements confirmed that DPI SiON and SiO2 surfaces have similar contact angles showing a good similarity of polarity [22] (link).
10
Multi-cellular Reporter Assay for ER-Mitochondria Dynamics
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NSC3450 (link), COS-751 (link), and HEK-29352 (link) cells were maintained at 37 °C and 5% CO2 in Dulbecco’s modified high glucose Eagle’s medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic (Thermo Fisher Scientific). For imaging, cells were plated on glass coverslips coated with poly-L-lysine (50 μg/ml) and bovine plasma fibronectin (10 μg/ml) (Life Technologies). Plasmid transfections were done using Lipofectamine 2000 (Invitrogen) in Opti-MEM media (Thermo Fisher Scientific). Cells were imaged or harvested 48 h post-transfection. V1-ER and V2-Mito reporter plasmids were transfected at a 1:1 ratio at a total DNA of concentration of 1 μg per well of a 12 well dish (except where otherwise indicated). MFN2 siRNA (Dharmacon) transfection was performed with lipofectamine RNAiMAX at an siRNA concentration of 50 nM for 48 h.
Stably transfected NSC34 cells co-expressing V1-ER and V2-Mito were selected on 400 μg/ml zeocin (InvivoGen).
ER stress was induced with tunicamycin (Calbiochem) at 2 μg/ml or 10 μg/ml for 6 h, with an equivalent volume of DMSO as vehicle only control.
Stably transfected NSC34 cells co-expressing V1-ER and V2-Mito were selected on 400 μg/ml zeocin (InvivoGen).
ER stress was induced with tunicamycin (Calbiochem) at 2 μg/ml or 10 μg/ml for 6 h, with an equivalent volume of DMSO as vehicle only control.
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