NPE cells were cultured to confluence on collagen IV-coated Flexible-bottomed BioFlex® 6-well culture plates (Flexcel International Corporation, Burlington, NC, USA). The BioFlex® 6-well plates were used with a BioFlex® Baseplate Kit and a Flexcell® Tension System (Flexcell® FX-5000TM) to provide a mechanical stretch protocol to NPE monolayers maintained at 37 °C in a humid atmosphere at 5% CO2 by placing the apparatus in a tissue culture incubator. The cells were subjected to 10% cyclic stretch at 0.5 Hz (30 cycles/min) for 1, 2, 5 or 10 min. Stretch and control (no stretch) experiments were carried out simultaneously using cells derived from a single pool grown on identical plates.
Fx 5000
Manufactured by Flexcell
Sourced in United States
The FX-5000 is a versatile bioreactor system designed for cell and tissue culture applications. It provides a controlled environment for the cultivation of cells, allowing for the manipulation of various parameters such as temperature, pH, and gas exchange. The FX-5000 is engineered to support a wide range of cell types and culture conditions, making it a reliable tool for researchers and scientists in the field of cell biology and tissue engineering.
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Lab products found in correlation
Collagen 1, by Flexcell (2 mentions)
Bioflex 6 well plate, by Flexcell (2 mentions)
Bioflex six well plate, by Flexcell (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Flex 1, by Flexcell (1 mentions)
Complete mouse endothelial cell medium, by Cell Biologics (1 mentions)
Bioflex collagen 1, by Flexcell (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Cfx connect system, by Bio-Rad (1 mentions)
16 protocols using fx 5000
1
Mechanical Stretch Stimulation of NPE Cells
2
Mechanical Loading on Endplate Chondrocytes
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The endplate chondrocytes were seeded into BioFlex six-well plates (Flexcell International Corporation, USA) for multidirectional mechanical loading. The Flexcell FX-5000TM strain loading system used a 0.5 Hz sinusoidal strain with 8%, 12%, and 16% elongation for 3 d, at 8 h/d. The cells were set as the loading group after intermittent tension stimulation by adjusting the softness and stiffness of the bottom wall of the six-pore plate to which endplate chondrocytes adhere. The cells in the control group were cultured normally.
3
Multiaxial Stretch Regulates Endothelial Cells
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Multiaxial stretching was performed using (1) a Flexcell FX-5000TM or (2) a custom device, as previously described66 (link)–68 (link). The same quantity of endothelial cells (2^105 cells/well) were cultured in six-well plates with silicone membrane surfaces coated with collagen type I prior to cell seeding. 100% confluent cells were exposed to low (2.5–5%) or high (12.5–15%) levels of multidirectional stretch to mimic venous or arterial conditions, respectively. It is demonstrated that SV graft exposed to the arterial pulsatile pressure (120/80 mmHg) receives a circumferential stretch of 10–15%8 ,10 (link),69 . Mechanical loading was performed at a 1 Hz loading frequency for up to 48 h. Additionally, cells without mechanical loading were cultivated in the same plate type and incubator as a control group. The culture media was not changed during experimental period. Some experiments were performed with concomitant chemical treatment, with no culture media changes and daily treatments with TGF-β2 and IL-1β, both at 10 ng/mL. All experiments were maintained at 37 °C in humidified air with 5% CO2.
4
Tensile Stress on Human Keratinocytes
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For FlexCell experiments, primary human keratinocytes were grown in BioFlex 6-well plates precoated with collagen I (FlexCell International) for KCs or plain BioFlex 6-well plates coated with fibronectin overnight prior to use (Sigma) for LECs. Cells were subjected to tensile stress at 15% effective stretch, 0.8 Hz, and half-sine waveform using the FlexCell FX5000 cell tension system (FlexCell International). Cells were stretched for 12 h and rested for a further 12 h prior to lysis. All cells were grown and subjected to tension in a humidified incubator at 37 °C with 5% CO2.
5
Biaxial Stretching of Cardiomyotubes
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Terminally differentiated H9C2 (myotubes) were stretched using the Flexcell FX-5000 strain unit (Flexcell International) that produces an isotropic two-dimensional (biaxial) strain of cells cultured on the flexible surface (silicone membrane) of the culture plates, as previously described [29 (link)]. Cardiomyotubes were subjected to four stretching protocols, in which the loading time was kept the same in each protocol, based on our previous findings [29 (link)], while the elongation and frequency of stretching varied: (a) 2% elongation (strain) at a frequency of 0.25 Hz; (b) 2% elongation at 1 Hz, (c) 12% elongation at 0.25 Hz, and (d) 12% elongation at 1 Hz. The waveform of the tension applied on the cardiomyocytes in the stretching cycle of each protocol mimicked the pressure fluctuations of a heartbeat in vivo.
6
Cyclic Stretch of Cells on Collagen
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A Flexcell FX-5000 strain unit (Flexcell International Corporation) was used in this study, with procedures similar to a previous study (16 (link)). Cells were cultured (5×105 cells/well) in a flexible bottomed polystyrene plate (6 wells) with type I collagen (0.15 mg/ml) coated at the bottom (Flex I, Bioflex Plates; Flexcell International Corporation). After cell attachment, cyclic stretch was applied to the cells at 37°C for different durations (0, 6, 12 or 24 h). Based on a previous study (16 (link)), the stretch parameter was set at a maximum 15% elongation. In the present study, the cultured cells were subjected to 15% elongation for 10 sec and then relaxation for the next 10 sec. In pathway inhibition tests, subconfluent cells were pre-treated with 0.5 µM Cyclopamine (Cpn; Sigma-Aldrich; Merck KGaA) for 2 h at 37°C and subsequently subjected to cyclic stretch for 24 h at 37°C.
7
Cyclic Mechanical Strain on Myoblasts
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Myoblasts were seeded on the elastic membrane of the Bioflex 6-well plate coated with Collagen I (Flexcell International Corporation, Burlington, NC, USA) at a cell density of 3 × 105 cells per well and were left to adhere overnight. The plate was placed on a circular shaped loading post of 25 mm in diameter. In the FlexCell FX-5000 tension system (Flexcell International Corporation, Burlington, NC, USA), the membrane was pulled downwards by vacuum suction which caused the membrane to stretch across the loading post. The cells received either 5% equibiaxial dynamic strain at a frequency of 1 Hz or constant, statical strain based on protocols from the literature [48 (link),50 (link)] and preliminary data. Cells were strained for 1 h followed by a rest period of 2 h. This loading was repeated for 3 times followed by a rest period of 4 h. One cycle took in total 13 h. The protocol was repeated for 24 h or 48 h.
8
Cyclic Stretch of Trabecular Meshwork Cells
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Cells were seeded at confluence on 6-well Flexcell plates (Flexcell International Corp., Burlington, NC) that were coated with type IV collagen-coated as previously described (Hauser et al., 2015 (link); Elliott et al., 2016 (link); Youngblood et al., 2020 (link)). Cells were incubated in 1% FBS DMEM 2 h prior to starting cyclic stretch. In vivo, TM cells are bathed and nourished in aqueous humor, which contains about 1% serum proteins (Freddo 2013 (link); Keller et al., 2018 (link)). Thus, in an attempt to simulate an in vivo environment, we chose to use 1% FBS instead of 0% FBS for use in experiments, which can unduly stress the cells. Cell stretch (20% stretch) was performed at frequency of 1 Hz, mimicking the ocular pulse, for 24 h in 1% FBS DMEM using the Flexcell FX-5000 (Flexcell International Corp., Burlington, NC). Evaluation of cells at 24-h was selected based on previous studies (Bradley et al., 2001 (link); Bradley et al., 2003 (link); Vittal et al., 2005 (link)).
9
Uniaxial Stretch Effects on MRECs
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Male and female MRECs were grown to confluency on six-well culture plates that permit uniaxial stretch (BF3011C, FlexCell, Burlington, NC). These cell culture plates were coated with collagen type I and 1% gelatin cross linked with 0.05% glutaraldehyde. The media were replaced every other day with complete mouse endothelial cell medium (Cell Biologics) and stored in a 37°C incubator with 5% CO 2 . Once confluent, uniaxial cyclical mechanical stretch was applied to the endothelial cell monolayer using a commercially available computer-driven strain unit (FX-5000, FlexCell). Cyclical stretch was applied for 48 h at 5%, 10%, and 15% elongation, 1 Hz, and ½ sine curve. MRECs were harvested from the stretch plates between 12 PM and 2 PM EST to have consistent circadian rhythm profiles in all groups.
10
Biaxial Strain Induces FAK Activation
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The biaxial strain was performed essentially as described elsewhere (Uzer et al., 2015 (link)). In brief, REF52 intact cells and cytoplasts were plated at a density of 30,000 cell/cm2 per well in six-well BioFlex collagen-I coated plates (BF-3001C; Flexcell). After plating for either 3 or 18 h, cells were subjected to dynamic uniform biaxial cyclic strain at 5% magnitude at 10 cpm for 20 min using the Flexcell FX 5000 under conditions of 37°C and 5% CO2. Control plates were handled the same but without strain application. Immediately after strain, whole-cell lysates were prepared and probed for phospho-FAK, total FAK, and GAPDH via Western blot analysis. Blots were analyzed using ImageJ. Data were derived from three independent experiments.
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