The cultured PDL cells were subjected to mechanical tensile loading in vitro using the Flexcell® 5000 tension system (Flexcell, Burlington, North Carolina, USA). In brief, cells were seeded onto flexible-bottomed BioFlex® (Flexcell) 6-well culture plates at a density of 2 × 105 cells/well and cultured until they reached 80% confluence. After a 12-hour period of serum starvation, the cells were subjected to static waves of 12% equibiaxial tensile strain using a computer-controlled vacuum stretch apparatus (17 (link)). PDL cells in the control group were cultured under the same conditions in the absence of tensile loading. Cells were subjected to tensile loading for 12 hours or 3 days for qPCR and western blotting, respectively, and continued to culture for 10 days prior to Alizarin Red S staining (ARS).
Bioflex
Manufactured by Flexcell
Sourced in United States
The BioFlex is a lab equipment product designed for cell culture studies. It facilitates the application of mechanical strain to adherent cells grown on flexible silicone membranes. The device allows for the controlled and quantifiable application of cyclic or static strain to study cellular responses to mechanical stimuli.
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Lab products found in correlation
Fx 4000t, by Flexcell (2 mentions)
Fx 5000 tension system, by Flexcell (2 mentions)
5000 tension system, by Flexcell (1 mentions)
Ldh detection kit, by Roche (1 mentions)
Fx 5000t tension system, by Flexcell (1 mentions)
Pt 2501, by Lonza (1 mentions)
Pt 3001, by Lonza (1 mentions)
Hmscs, by Lonza (1 mentions)
Fx 3000 strain unit, by Flexcell (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
Fx 3000 tension system, by Flexcell (1 mentions)
Flexercell strain unit fx 3000, by Flexcell (1 mentions)
Flexercell fx 5000 strain unit, by Flexcell (1 mentions)
Strain unitfx 5000, by Flexcell (1 mentions)
Fx 4000 tension system, by Flexcell (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
2 bromopalmitate, by Merck Group (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
Palmostatin b, by Merck Group (1 mentions)
24 protocols using bioflex
1
Mechanical Loading Regulates PDL Cell Mineralization
2
Mechanical Stretch Induces Metabolic Adaptation
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Human arterial smooth muscle cells (HUASMCs) were freshly isolated from individual umbilical cords and grown on collagen I-bonded BioFlex® plates (Flexcell International, Hillsborough, NC, USA) with DMEM medium containing 15% FCS (osmolarity: 305 mosm ± 4.08 mOsm). The isolation of HUASMCs was approved by the local Ethics Committee (Heidelberg, Germany; reference 336/2005) and conformed to the principles outlined in the Declaration of Helsinki (1997). Stretch was typically applied at a frequency of 0.5 Hz and an elongation of 0–13% for 24 h by using a Flexercell FX-5000 tension system. In addition, cells were exposed to the following reagent: 40 μmol/L Etomoxir (Calbiochem, Germany). The compound was dissolved in DMSO. Pure DMSO (0.4%, v/v) was simultaneously applied to the control cells as solvent control.
3
Simulating Elevated Intraocular Pressure
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To explore a proper strain parameter that represented the in vivo elevated IOPs, we attempted to apply multiple mechanical strains by the Flexcell FX‐5000™ tension system (Flexcell International Corporation). The ppHSFs were seeded into the six‐well collagen‐I coated Bioflex plates (Flexcell International Corporation), with 2.5 × 105 cells per well. After the cells were attached to the plates under 24‐h culturing with conventional serum‐containing DMEM, the media were changed with serum‐free DMEM for 24 h, then DMEM containing 1% FBS before the biaxial strain. In the present study, we applied 0, 5% 0.5 Hz, 10% 0.5 Hz, 20% 0.5 Hz strain for 8 and 24 h, respectively.
4
Evaluating Neuronal Membrane Integrity
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The lactate dehydrogenase (LDH) detection kit (Roche) was used to evaluate cell membrane integrity. The primary neurons at 24 hours post-SI were cultured in six-well BioFlex® culture plates (Flexcell International Corp.). The amount of LDH released into the culture medium is represented as LDH activity. The optical density was read at 490 nm with a spectrophotometer (BioTek).
5
Cyclic Stretch of Cells on Collagen
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Cells were seeded on collagen-coated 6-well BioFlex plates (Flexcell International Corporation) at a density of 30,000–50,000 cells per well. Cells were left to adhere overnight, and then stretched using a Flexcell FX-5000 Tension unit for 24 h with 10% uniaxial cyclic stretch at a frequency of 0.5 Hz.
6
Tensile Strain Device for HPDLC Mechanobiology
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To mimic the tensile force exerted on HPDLCs during OTM, a custom-made strain device Tension Plus System, was used [19 (link)]. The tension system has been further modified in the present study by positioning six planar faced posts beneath the silicon membranes of the culture wells to provide the uniform strain to the cultured cells. Equal numbers of HPDLCs were seeded into the six-well, flexible-bottomed plate coated with type I collagen (BioFlex, Flexcell International Corp, Hillsborough, NC). After reaching 80% confluence, the medium was refreshed and cyclic tensile force (10% bottom membrane deformation) was applied to HPDLCs at a frequency of 0.1 Hz (5 s stress and 5 s rest) for 24 h for mRNA extraction and 72 h for ALP staining, western blots analyses and immunofluorescence detections. HPDLCs that were maintained in identical conditions without being stretched were used as control. After stretch application procedure, cells were harvested for protein or mRNA expression analyses or were subjected to ALP staining assays.
7
Mechanical Stretch of C2C12 and L6 Myoblasts
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C2C12 and L6 myoblasts were stretched as described in our previous studies[17 (link), 26 (link)]. Briefly, cells were plated onto flexible-bottomed 6-well plates pre-coated with type-I collagen (BioFlex, FlexCell International Corporation, USA) at a density of 1 × 105/well density and incubated for 24 h before exposing to mechanical strain. Cells were then subjected to cyclic mechanical stretch of 15% or 20% elongation at 0.5 Hz frequency for 6 h using a computer-controlled vacuum stretch apparatus (FX-5000 T Tension System, FlexCell International Corporation). In parallel with identical experimental conditions, cells grown in flexible-bottomed 6-well plates but left un-stretched were considered as control.
8
Murine MSC Osteogenesis and Stretch Response
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Murine MSCs (C3H10T1/2, ATCC, CCL-226) were seeded using growth medium composed of Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). For inducing osteogenesis, growth medium was replaced by osteogenic differentiation medium (further containing 10 mM β-glycerophosphate, 10 nM dexamethasone, and 50 μg/mL ascorbic acid) when cells reached confluence. The medium was changed every 2-3 days. MSC osteogenesis was evaluated by qRT-PCR and western blotting of osteogenic transcription factor and genes on day 7 (after giving osteogenic media) and Alizarin red staining of synthesized bone minerals on day 21. The key experiment of YAP nuclear translocalization under stretch loading was repeated with human mesenchymal stem cells (hMSCs, Lonza, PT-2501, 25 year-old female) using mesenchymal stem cell growth medium (Lonza, PT-3001). All experiments throughout this study were done on type-I collagen-coated cell stretch plates (Flexcell International, BioFlex).
9
Mechanical Stretch Regulates LPAR and LPP Expression
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Primary cultures of HTM cells were plated onto BioFlex six-well plates precoated with collagen type I (Flexcell International Corp., Burlington, NC, USA). As the cells reached confluence, cultured medium was switched to serum-free, phenol-free DMEM, and cells were subjected to cyclic mechanical stretch (15% stretching, 1 cycle/sec) for 24 hours, using the computer-controlled vacuum-operated FX-3000 Flexcell Strain Unit (Flexcell, Hillsborough, NC, USA), as we previously described.10 (link) Control cells were cultured under similar conditions with no mechanical force applied. RNA was extracted from the cells and analyzed by RT-quantitative (q)PCR to monitor changes in level of expression of LPARs, LPPs, and ATX.
10
Rat Myoblast Culture and Passaging
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L6 rat myoblasts (The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences) were cultured in a humidified incubator at 37°C in an atmosphere containing 5% CO2. Cultures were grown in DMEM (HyClone; GE Healthcare Life Sciences), containing 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences), 100 U/ml penicillin and 100 µg/ml streptomycin. Growth medium was replaced every other day. When the cells reached 80% confluence, they were digested with 0.25% trypsin and passaged at a split ratio of 1:2. Cells were then seeded in growth medium into each well of a 6-well plate (BioFlex; Flexcell International Corporation) at 3×105 cells/well.
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