Commercial stretching chambers (10 cm2, STB-CH-10, STREX, Taiwan) were coated with 50 µg/mL fibronectin (FC010, Merck Millipore, Darmstadt, Germany) overnight at 37 °C. The human AF cells were seeded in the chambers (100,000 cells per chamber in 5 mL growth medium) and cultured for 72 h at 37 °C, 5% CO2, unless otherwise stated. The cells were serum-starved for 12 h in a serum-free medium. The chambers were mounted on a commercial stretching bioreactor (STB-140-10, STREX, Taiwan) and subjected to 5% (light cyclic stretch, LCS) or 15% (high cyclic stretch, HCS) cyclic sinusoidal uniaxial strain at a frequency of 1 Hz at 37 °C and 5% CO2. Control chambers were kept in identical conditions without stretching. Cells were stretched for different amounts of time. The stretching started with the chambers that were stretched for 24 h, and shorter duration chambers were added progressively on the device so that the stretching was stopped at the same time for all conditions. Immediately after the mechanical loading, the cells were lysed for gene expression analysis.
Fc010
Manufactured by Merck Group
Sourced in United States, Germany
The FC010 is a laboratory equipment designed for centrifugation purposes. It is a compact, versatile, and reliable centrifuge that can be used to separate various biological samples. The device features a simple and intuitive control panel, allowing users to easily set and monitor the operating parameters.
Automatically generated - may contain errors
Lab products found in correlation
German glass coverslips, by Electron Microscopy Sciences (4 mentions)
L2020, by Merck Group (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Bp180, by Thermo Fisher Scientific (3 mentions)
Cc095, by Merck Group (2 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (2 mentions)
Fx 6000t, by Flexcell (2 mentions)
Fibronectin, by Flexcell (2 mentions)
Fibronectin, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Pelco easiglow, by Ted Pella (2 mentions)
Hepes buffer, by Corning (2 mentions)
C5533, by Merck Group (2 mentions)
Alexa fluor phalloidin, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic anti anti, by Thermo Fisher Scientific (1 mentions)
Collagenase nb4, by Serva Electrophoresis (1 mentions)
Dispase 2, by Roche (1 mentions)
F7524, by Merck Group (1 mentions)
Mab1976, by Merck Group (1 mentions)
50 protocols using fc010
1
Cyclic Stretching of Human AF Cells
2
Deformable PAA Gel Substrate Preparation
3
Cell Spreading on Extracellular Matrix Proteins
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The wild type or PINCH-1 KO A549 cells were allowed to adhere and spread on glass coverslips precoated with collagen Ɩ (20ug ml−1, Corning, #354249), laminin (20ug ml−1, Sigma-Aldrich, L2020) or fibronectin (20 ug ml−1, EMD Millipore, FC010) in a cell culture incubator at 37 °C in the presence of 5% CO2 for 2 h. The coverslips were washed three times with PBS, and the adhered cells were fixed with 4% paraformaldehyde and stained with Alexa Fluor–phalloidin (Life Technologies). The area of cell spreading was quantified using Image J software (version 2.0.0-rc-69/1.52p, NIH, Bethesda, MD, USA).
4
Deformable PAA Gel Substrate Preparation
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Deformable PAA gel substrates as described previously 1 (link),30 (link),42 . Briefly, cover slips were glued into custom-made petri dishes, cleaned and silanized with (3-aminopropyl)trimethoxysilane (APTMS; unless otherwise stated, all chemicals from Sigma-Aldrich, Dorset, UK) for 3 min (minutes), treated with glutaraldehyde (diluted 1:10) for 30 min. Gel premixes were made by thoroughly mixing 440 μL of 40% acrylamide, 60 μL of 100% hydroxyl-acrylamide, and 250 μL of 2% Bis-acrylamide. The premix was then mixed with PBS at ratios between 40 μL to 460 μL and 150 μL to 350 μL to achieve gel stiffness between 50 Pa and 20 kPa. Polymerization was initialized by adding 0.3% (v/v) N,N,N′,N′-tetramethylethylenediamine (TEMED) and 0.1% (w/v) ammonium persulfate (APS) and 20 μL of the solution (giving about 100 μm gel thickness) were covered with cover slips, which were made hydrophobic with RainX (Shell Car Care International Ltd, UK) for 10 min beforehand. After at least 20 min top cover slips were removed, washed 2x with PBS, sterilized under UV light for 30 min, functionalized with either 100 μg/mL poly-D-lysine overnight for microglia cells or with 0.2 mg/mL fibronectin (FC010, Merck, 1:5 in PBS) for 2h at 37°C for fibroblasts, and washed 2x with PBS.
5
Sprouting Angiogenesis Assay in Microfluidics
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To perform the sprouting angiogenesis assay, the cell culture channel is first treated with 50 μl of ECM protein, fibronectin (FC010, Merck KGaA, Darmstadt, Germany), with a concentration of 0.1 mg/ml for 2 h at 37 °C to promote the cell adhesion after the hydrogel matrix formation. The fibronectin solution is replaced by the complete growth medium after the treatment, and the HUVECs (30 μl with a cell density of 107 cells/ml) are then seeded into the cell culture channel. The HUVECs are cultured for more than 3 h in the devices before applying the oxygen gradients, and the entire cell experiments are performed for more than 72 h.
6
Isolation and Expansion of Bovine Nucleus Pulposus Cells
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Tails from 18 to 24-month-old cows were obtained from the local slaughterhouse and carefully dissected to expose the IVDs. Nucleus pulposus (NP) biopsies were minced and digested overnight at 37°C, 5% CO2, using 0.4% collagenase NB4 (17454.01, Serva) and 0.2% dispase II (04942078001, Roche) dissolved in PBS 1X with 5% Antibiotic-Antimycotic (Anti-Anti; 15240-062, Gibco). The next day, the tissue digest was filtered through a cell strainer and the cell suspension was centrifuged at 1000 rpm for 5 min. The pellet was washed with culture medium (Dulbecco’s modified Eagle medium/F-12 Nutrient Mixture (DMEM/F12; 31330-038, Gibco), 10% fetal calf serum (FCS; F7524, Sigma-Aldrich) and 1% Anti-Anti). After centrifugation and resuspension in culture medium, cells were seeded in fibronectin-coated (0.01 mg/mL human plasma fibronectin; FC010, Merck Millipore) culture flasks (150 cm2). Cells were expanded to passage 1–2 at 37°C, 5% CO2, and the medium was changed twice per week.
7
Visualizing LTBP-1 Binding to Myofibroblasts
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To visualize direct binding of cells to purified LTBP-1, we used microcontact printing (Goffin et al., 2006 (link)). In brief, polydimethylsiloxane stamps exhibiting islet topographies with dimensions of 10-µm length, 1.5-µm width, and 4-µm spacing were coated with 0.2 µg/ml LTBP-1 or FN (FC010; EMD Millipore) for 1 h. Protein prints were created on plastic coverslips, and protein-free areas were passivated with poly-l -lysine–polyethylene glycol for 10 min. Myofibroblasts were seeded on prints in serum-free media for 4 h before being processed for immunostaining. For cell adhesion quantification, tissue culture plastic wells were coated with 20 µg/ml LTBP-1, and hDMfs were seeded for 4 h in the presence of cyclic peptides antagonizing 10 µM RGD (12135-010; Gibco), 10 µM control RGE (12139-010; Gibco), integrins αvβ3/αvβ5 (EMD121974; Cilengitide), αvβ3 integrin (EMD66203), and scrambled control (EMD135981; Merck). Blocking antibodies were used directed against 10 µg/ml integrins β1 (MAB1965; EMD Millipore), β3 (MAB1976; EMD Millipore), and β5 (MAB1961; EMD Millipore). Additional controls were 10 µM RGE and human IgG (I9135; Sigma-Aldrich). Samples were rigorously washed three times before cells were fixed for quantification and staining.
8
Cyclic Stretching of Human AF Cells
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Commercial silicone stretching chambers (4 cm2, STB-CH-10, STREX) were passively coated with 50 µg/mL fibronectin (FC010, Merck Millipore, MA, USA) at 37 °C for 24 h. The human AF cells at passages 5–6 were seeded in the silicone chambers (1 × 105 cells per chamber in 5 mL growth medium) and cultured at 37 °C, 5% CO2 for 3 days, unless otherwise stated. The cells were cultured in a serum-free medium for 12 h before being applied to stretching apparatus. The AF cells cultured in chambers were mounted on a commercial stretching bioreactor (STB-140-10, STREX) and subjected to 5% (light cyclic stretch, LCS) or 15% (high cyclic stretch, HCS) cyclic sinusoidal uniaxial strain at60 cycle/min at 37 °C and 5% CO2. Cells were stretched for designated time courses and time-matched control chambers were kept in static conditions without stretching. Immediately after the mechanical loading, the cells were lysed for gene and protein expression analysis.
9
Adherent HEK-293T Cell Fixation
10
Visualization of Mitochondrial C1qbp and Superoxide
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Cells were grown on chamber slides precoated with fibronectin (Sigma, FC010). The cells were washed 3 times with phosphate buffer saline (PBS) and then fixed with 4% paraformaldehyde for 15 min; after three times washes with PBS, the samples were blocked with 1% BSA in PBS containing 0.1% Triton X-100(PBS-T) and then incubated with primary antibodies at 4 °C overnight. The next day, after incubation with corresponding secondary antibody and Hoechst 33,342 at room temperature for 2 h, the samples were washed in PBS. Cells were loaded with 200 nM Mitotracker (Beyotime, C1035) for 30 min in a humidified incubator with 5% CO2 at 37 °C. After fixation, permeabilization, and blocking, cells were then incubated with anti-C1qbp overnight at 4 °C. DHE (Vigorous Biotechnology Beijing Co, Ltd, R001) was used to detect intracellular superoxide. DHE (5 μM) was added to cells and incubated at 37 °C for 90 min in the dark. At the end of the incubation time, cells were washed with PBS. Images were captured under the Zeiss LSM800 confocal microscope. Use ImageJ software to analyze cells.
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