Static stretch was administered (FX-6000 Tension System, Flexcell International Corporation) as previously described (Hornberger et al., 2005 (link); Baccam et al., 2019 (link)) with the following modifications. Cells were subjected to a static 5% uniaxial stretch in the last 4 or 24 h of CM treatment (as described above). Control cells (0% stretch) were grown under identical conditions but left unstretched and placed beside the incubator’s baseplate. For administration of stretch, culture plates were removed from the incubator, rapidly placed onto the stretch device’s baseplate, and then placed back in the incubator. The Flexcell Arctangle® loading station was used for all experiments, which consists of six rigid posts covered in a thin layer of silicone lubricant, centered beneath the six-well Uniflex plate. Applied vacuum pressure deforms the membrane across the loading post in a single direction (e.g., north and south poles), creating a uniaxial stretch (Figure 3B ). A custom ramp protocol, described here, was designed to ensure cell adherence to the membrane while vacuum pressure is applied: the Flexcell tension system was set to increase vacuum pressure gradually, and thus % elongation in increments of 1% every 2 s, up to 5%, which is then maintained for the duration of the experiment (e.g., 24 h), followed by a ramp-down to 0% at the conclusion of the experiment.
Fx 6000 tension system
Manufactured by Flexcell
Sourced in United States
The Flexcell-FX-6000-Tension System is a laboratory device designed to apply controlled cyclic or static tension to cell cultures. The system utilizes a computer-controlled vacuum pressure system to stretch and deform flexible-bottomed culture plates, subjecting the cells to a defined mechanical environment.
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11 protocols using fx 6000 tension system
1
Uniaxial Stretch Protocol for Cell Culture
2
Mechanical Stretching of HTM Cells
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HTM cells were cultured in collagen-coated plates (Flexcell International, Burlington, VT, USA, BioFlex plates, BF-3001C), and after 90% confluency, the plates were fixed on a FlexCell (Flexcell International, FX-6000 Tension System). The cells were stretched at 0.69 Hz frequency with 15% stretching for 8 h for gene expression analysis and 24 h for protein expression analysis based on previously published reports [65 (link),66 (link),67 (link),68 (link)]. Cells and media were collected and processed, as mentioned later, for RNA analysis and immunoblotting.
3
Mechanical Stretching of PDLSCs
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The PDLSCs prior to the fifth passage were seeded onto collagen I-coated, amino silicone-bottomed 6-well cell culture plates (Flexcell International, USA) at a density of 2.0 × 105 PDLSCs per well. Cells were cultured under the standard conditions described above. After reaching ~ 80% confluence, PDLSCs were subjected to serum deprivation at 2% FBS (Lonsera, China) for 24 h. The cells were then mechanically stretched with the FX-6000 Tension System (Flexcell International, USA) at 10% elongation and 0.5 Hz. The control group of cells was cultured under the identical condition as above, but without applying the mechanical stimuli.
4
Mechanical Stretch-Induced Lung Injury Model
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We performed an additional experiment to investigate the roles of EphA2 and NOX4 in alveolar macrophages. The J774A.1 cell line obtained from the ATCC (TIB-67™) was used in a mechanical stretch-induced lung injury model. J774A.1 cells were cultured at a density of 1 × 106 cells/cm2 in DMEM + 2 mM glutamine + 10% fetal bovine serum. We used an in vitro mechanical ventilation model using cyclic stretching of alveolar macrophages to mimic high and low tidal volume ventilation strategies. Cells were biaxially stretched in a triangular wave manner for 2 or 4 h at 37 °C using the FX-6000 Tension System (Flexcell, Burlington, NC, USA). Thirty cycles/min, with a stretch/relaxation relation of 1:1, and 20% changes in the basement membrane surface area were applied for mechanical stretching.
We measured the levels of IL-6 (Koma Biotech, catalog no. K0331230HS, Seoul, South Korea) and IL-8 (Invitrogen, catalog no. EMCXCL1, Waltham, MA, USA) with and without EphA2 (10 µM) or NOX4 (10 µM) inhibition. We subjected the macrophages to the following treatments:
We measured the levels of IL-6 (Koma Biotech, catalog no. K0331230HS, Seoul, South Korea) and IL-8 (Invitrogen, catalog no. EMCXCL1, Waltham, MA, USA) with and without EphA2 (10 µM) or NOX4 (10 µM) inhibition. We subjected the macrophages to the following treatments:
No treatment (control).
Mechanical stretch for 2 or 4 h.
EphA2 inhibition (1 h before cell stretch) + Mechanical stretch.
NOX4 inhibition (1 h before cell stretch) + Mechanical stretch.
5
Mechanical Stretching of HTM Cells
6
Mechanical Stimulation of PDLSCs
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The Flexcell® FX-6000™ Tension System (Flexcell International Corporation, Burlington, NC, USA) was utilized to apply mechanical stimuli. Briefly, PDLSCs were seeded on Flexcell Amino silicone-bottomed plates at 2.0 × 105 cells/well with α-MEM, containing 10% fetal bovine serum. When the cells reached 80% confluence, the above plates were placed into the tension system, and an 8% stretch at 0.5 Hz was applied for 24 h. Meanwhile, plates without stress were set as the negative control group.
7
Cyclic Mechanical Stimulation of HTM Cells
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Up to four biological replicates of HTM cells were seeded (∼40,000 cells/well) in collagen-coated plates (BioFlex plates, BF-3001C Flexcell International, Burlington, NC, United States) after counting using a hemocytometer. Once cells attain 90% confluency, cells were serum-starved for 3 h. Plates were placed on a Flexcell FX-6000 Tension System (Flexcell International, NC, USA). The cells were subjected to CMS at 0.69 Hz frequency with 15% stretching as published earlier (Luna et al., 2009 (link); Shim et al., 2020 (link); Youngblood et al., 2020 (link)) for 8 h for mRNA sequencing (HTM lines 1–4) and 24 h for proteomics and lipidomic analyses (HTM lines 1, 2, and 4). Control cells were cultured in the same conditions but were not subjected to CMS.
8
Mechanotransduction of MscL-G22S in SCs
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To investigate the effects of mechanical stretching-induced MscL-G22S, four groups of SCs with different treatments were set as follows, MscL-G22S-expressing SCs with mechanical stretching (MscL-G22S activation), MscL-G22S-expressing SCs without stretching, SCs with mechanical stretching, and SCs without treatment (neither stretching nor MscL expression). The SCs were seeded in a BioFlex® six-well plate (Flexcell International Corporation, NC, USA) with laminin-precoated elastic membranes for the stimulus of mechanical stretching at 0 ~ 6% strain and 0.2 Hz for 24 h using the Flexcell® FX 6000™ Tension System (Flexcell International Corporation, NC, USA) inside the regular incubator (37 °C, 5% CO2). For the un-stretched controls, SCs seeded in the same BioFlex® plates were maintained in an identical manner but in the absence of any membrane tension. After a culture of 24 h, the cell viability was measured in a microplate reader (BioTek Cytation Hybrid, Agilent, CA, USA) using cell counting kit-8 (CCK-8, Dojindo, CK04, MD, USA) according to the manufacturer’s instructions. To investigate the role of PI3K in the action mechanism of MscL-G22S activation on SCs, a potent inhibitor of PI3K, LY294002 (MEC, HY-10108, NJ, USA) was added into the culture of MscL-G22S-expressing SCs (10 mM) before mechanical stretching in some experiments.
9
Mechanically-Induced PDLSCs Stretching
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Mechanical force was applied using the Flexcell-FX-6000-Tension System (Flexcell International Corporation, Burlington, NC USA). PDLSCs were seeded onto excell Amino silicone-bottomed plates 6 well cell culture plates coated with Collagen I solution (Collagen I, rat Tail, Corning, NY, USA) at a density of 2.0×10 6 cells per well. After the density reached ~80% con uence, the cells were serum deprived (2% serum) for 24 h before stretching. We imposed 10% stretch at 0.5 Hz. The control group was cultured in same silicone bottomed plates and the same culture environment without stretching.
10
Mechanical Stretch Effects on ABMMCs
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Flexcell amino silicone rubber plates coated with collagen type I (Collagen I, rat tail, Corning, NY, USA) were used to culture ABMMCs. Cells were subjected to cyclic mechanical stretch (10% elongation, 0.5 Hz; sinusoidal waveforms) for 6, 12, and 24 h by the Flexcell-FX-6000-Tension System. The control group was cultured under the same conditions without stretching.
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