Chondrocytes were grown on collagen I‐coated Bioflex 6‐well culture plates (Flexcell International, Hillsborough, NC) to 80%‐90% confluence. CTS experiments were performed using the FX‐5000 Flexcell system (Flexcell International, McKeesport, PA). To provide uniform radial and circumferential strain on the membranes, the plates were placed on a loading station (located in an incubator with 5% CO2) such that when a vacuum was applied to the loading station, the membrane deformed across the post face, creating uniform biaxial strain. Chondrocytes were subjected to CTS (10%, 0.5 Hz) for different durations (0, 0.5, 1, 2, 4, 8, and 16 hr) with or without IL‐1β for 24 hr. The stimulations of CTS and IL‐1β on chondrocytes began at the same time. We choose the best condition for further study. Compound C (ab120843; Abcam), a selective and reversible AMPK inhibitor, was used for pretreatment for 1 hr before the stimulation with IL‐1β and CTS (Dai et al., 2017 ).
Fx 5000 system
Manufactured by Flexcell
Sourced in United States
The Flexcell FX-5000 system is a versatile bioreactor platform designed for cell and tissue culture applications. It provides a controlled mechanical environment for the in vitro cultivation and study of various cell types. The system utilizes computer-regulated, pneumatic-based technology to apply precise and programmable mechanical stimuli to cultured cells.
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Lab products found in correlation
Bioflex 6 well culture plates, by Flexcell (2 mentions)
Ab120843, by Abcam (1 mentions)
Matlab, by MathWorks (1 mentions)
5 aza 2 deoxycytidine 5 aza, by Merck Group (1 mentions)
Bioflex plate, by Flexcell (1 mentions)
Complete ecm medium, by ScienCell (1 mentions)
Collagen 1 coated silicone membrane plates, by Flexcell (1 mentions)
13 protocols using fx 5000 system
1
Biaxial Strain on Chondrocytes
2
Chondrocyte Isolation and Cyclic Tensile Strain
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Donor chondrocytes were isolated as previously described41 (link). The chondrocytes were at passage 2 and fibroblasts were used as control. We detect specific markers in both cells by immunofluorescence analysis. The results showed that the expression of Sox9 is high in our chondrocytes, while low in fibroblasts (Supplementary Figure 1A ). Meanwhile, expression of S100A4 is low in our chondrocytes, while high in fibroblasts (Supplementary Figure 1B ). Chondrocytes (5 × 105/well) were grown on pronectin-coated Bioflex six-well culture plates (Flexcell International, Hillsborough, NC) to 80% confluence. Cyclic tensile strain (CTS) experiments were performed using the FX-5000 Flexcell system (Flexcell International, McKeesport, PA). CTS was enforced at 5%, 10%, and 15% elongation (0.5 Hz) for 24 h42 (link)43 (link).
3
Chondrocyte Response to CTS and TRAIL
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Chondrocytes were grown on collagen I-coated BioFlex 6-well culture plates (Flexcell International, Hillsborough, NC) to 80% to 90% confluency. CTS experiments were performed using the FX-5000 Flexcell system (Flexcell International, McKeesport, PA). The plates were placed on a loading station in an incubator with 5% CO2 such that when the vacuum was applied to the loading station, the membrane deformed across the post face, creating uniform biaxial strain. Chondrocytes were subjected to CTS (10%, 0.5 Hz) for 0, 1, 2, 4, 8, or 12 h in the presence of 100 ng/ml TRAIL. The stimulation of CTS and TRAIL on chondrocytes began at the same time. A 4 h treatment with CTS was determined to be the optimal duration and was used for subsequent experiments. All chondrocytes were harvested at 12 h with TRAIL.
4
Mechanical Strain on Cell Morphology
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Mechanical straining experiments were performed using FX-5000 Flexcell system (Flexcell Corp. (Mc-Keesport, PA)). Cells were seeded on the microposts at a density of 2500 cells/cm2 and were allowed to adhere overnight before the strain experiment (24 hours). The Uniflex membranes bound to the microposts were uniaxially and cyclically stretched (10%, 0.5 Hz) for 19 hours. The total duration of the experiment, from seeding to fixation, was 33 hours. Strain fields on the elastomeric microposts were characterized within the central region of the Flexcell membrane on which the microposts were bound by use of a Matlab-based (Mathworks Inc., Natick, MA) digital image correlation (DIC) code. A random pattern of fiducial markers was inked on the microposts bonded to the FlexCell membrane and images were recorded by mounted on a Zeiss stereomicroscope. By applying our uniaxial straining protocol (10%, 0.5 Hz, sine wave), results showed that in the area under consideration the strain field covers a range between 5% and 7%. Strains in the x direction (direction of applied strain) are 6.8% on average, and strains in y direction show a compression of 2%. Cells under static conditions grown for 33 hours on microposts bonded to glass coverslips were used as control.
5
Optimizing Synovial Macrophage Responses to Cyclic Tensile Strain
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Synovial macrophages were grown on bronectin-coated Bio ex six-well culture plates (Flexcell International, Hillsborough, NC, USA) to 80-90% con uence. CTS experiments were conducted using the FX-5000 Flexcell system (Flexcell International, McKeesport, PA, USA). To provide uniform radial and circumferential strain on the membrane, plates were placed in a loading station (in a 5% CO 2 incubator), such that when a vacuum was applied, the membrane deformed across the post face, creating a uniform bi-axial strain. Synovial macrophages were subjected to CTS (10%, 0.5 Hz) of different durations (0, 6, 12, 24, 48, 72, or 96 h), and LXA 4 concentrations (0, 0.5, 1, 2, or 4 nM) for 24 h, following the methods of our previous study [11] (link). After these steps, we selected the optimal conditions for further study (Fig. 2B).
6
Cyclic Strain on Cardiac Progenitor Cells
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Cyclic strain was applied using a FX-5000 Flexcell system (Flexcell International Corporation). Undifferentiated and predifferentiated L9TB CMPCs were seeded on collagen IV-coated Bioflex plates. Cells were seeded at 75% confluency and left for 24 h to allow attachment to the membrane. Uniaxial cyclic strain was applied by stretching the membranes on rectangular posts oriented in the x direction (901, Fig. 1B andD). Strains of 10% (0.5 Hz, sine wave) were applied for up to 48 h (day 2) in the y direction (01, Fig. 1B andD).
Samples were analyzed at day 0 (before the onset of the straining protocol), after 24 h (day 1), and 48 h (day 2) of cyclic straining.
Unstrained samples, cultured on identical Bioflex plates and under the same conditions, were used as controls.
Samples were analyzed at day 0 (before the onset of the straining protocol), after 24 h (day 1), and 48 h (day 2) of cyclic straining.
Unstrained samples, cultured on identical Bioflex plates and under the same conditions, were used as controls.
7
Biaxial Strain Stimulates FAK Phosphorylation
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Cells were plated onto 6-well Bioflex plates with silicone bottoms (Flexcell International, Hillsborough, NC). Uniform 2% biaxial strain was delivered at 10 cycles per minute for 20 minutes using the Flexcell FX-5000 system (Flexcell International, Hillsborough, NC). Controls were handled the same way but were not strained. Following strain, Focal Adhesion Kinase (FAK) phosphorylation at Tyr 397 site was measured as we have previously reported39 (link).
8
Cyclic Tensile Stress Regulates Fibroblast Behavior
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Cells were plated at a density of 1 × 105 cells per dish on collagen I-coated silicone membrane plates (Bioflex, Flexcell International, NC, the USA) and cultured for 2 days before beginning experiments. Cyclic tensile stress was applied to fibroblasts plated on six-well Bioflex Collagen I-coated plates using the Flexcell FX-5000 system (Flexcell International). A regimen of 10% tensile strain was delivered at 0.5 Hz for 2 h each day. Cells used for CCK8 and ALP (alkaline phosphatase) staining, Western blotting, qRT-PCR and immunofluorescence were processed immediately after application of cyclic tensile stress. Cells cultured under similar conditions but without cyclic stretch were used as unstretched controls.
9
Culturing and Stretching Human Pulmonary Artery ECs
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Human pulmonary artery ECs were obtained from Lonza (Walkersville, MD) and cultured as described previously [28 (link),29 (link)] in the manufacturer’s recommended endothelial growth medium-2 (EGM-2). Cells were grown at 37°C in a 5% CO2 incubator, and passages 6–9 were used for experiments with media changed 1 day before experimentation. For cyclic stretch (CS) studies, ECs were plated on Bioflex collagen I type cell culture plates (FlexCell International, Hillsborough, NC) and stimulated for 4 h at 18% CS as previously described [30 (link)] on the FlexCell FX-5000 System (FlexCell International), mimicking high tidal volume ventilation. For demethylation studies, ECs were treated with 5-aza-2′-deoxycytidine (5-Aza) (Sigma–Aldrich, St. Louis MO) for 24 h at indicated concentrations to inhibit DNA methyltransferase enzymes as described previously [31 (link)].
10
Mechanical Stretch of Lung Endothelial Cells
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Primary lung microvascular endothelial cells (LMEVCs) were extracted from male neonatal C57BL/6N mice (3 days old) using a tissue block attachment method (Additional file 1 : Supplementary content). Complete ECM medium (ScienCell, USA) containing 5% fetal bovine serum, 1% triple antibiotics, and endothelial cell growth factors was used. Cells were identified by immunocytochemistry with the endothelial marker CD31. Third-generation LMVECs were inoculated into a 6-well Bioflex plate (Flexcell, USA). Cells were subjected to cyclic mechanical stretch (MS) in the Flexcell FX-5000 system using the following parameters: 0.5 Hz (30 times/minute); 20% max elongation; 4-h duration. After modeling, the cells were treated with rFGF21 or phosphate-buffered saline (PBS), transferred to a conventional incubator (37 °C, 5% CO2), and cultured for 24 h before subsequent experiments.
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