Fibroblast culture and strain
Primary human dermal fibroblasts (#PCS-201-012) were purchased from American Type Culture Collection (ATCC, Manassas, Virginia, USA) and cultured as directed by the vendor. Cells were free of mycoplasma contamination as confirmed by the MycoProbe Mycoplasma Detection Kit (R&D Systems, Minneapolis, Minnesota, USA) used as directed by the manufacturer. Using a parallel study design as detailed in Figure 1A and Figure 2A, cells were combined from separate flasks then seeded at 120,000 cells per well on 6-well flexible collagen I-coated membranes (Flexcell International, Burlington, North Carolina, USA). The next day, cells were left unstrained (control) or mechanical stimulation was performed on a Flexcell FX-6000 according to two previously reported strain profiles . Briefly, for the first cyclic short-duration strain (CSDS) profile, fibroblasts were subjected to an 8-hour cycle with 1.6 second bouts of deformation increasing at 33.3%/second starting from rest to a maximum of 10% beyond resting length, followed by decreasing strain to baseline at 33.3%/second (Figure 1A). For the second CSDS profile, fibroblasts were subjected to an 8-hour cycle with 1.6 second bouts of deformation increasing at 22%/second starting at a baseline strain of 10% and a maximum of 16.6%, followed by decreasing strain to baseline at 22%/second (Figure 3A). For acyclic long-duration strain (ALDS), after a 3-hour rest period following CSDS, cells were subjected to a single 60 second bout of stretch at 6% beyond resting length at a loading rate of 3%/second followed by release at 1.5%/second until return to resting length. Conditioned media was collected simultaneously from all samples either 24 hours or 96 hours after initiation of the CSDS strain protocol and stored at -80°C.
IL-6 and IL-8 Enzyme-Linked Immunoassays
The collected conditioned media was analyzed using ELISA kits for human IL-6 and IL-8 (ThermoFisher Scientific, Waltham, Massachusetts, USA) to determine the concentration of these respective proteins. The ELISA methods were performed following instructions provided by the manufacturer and quantified on a FluoStar OPTIMA (BMG, Cary, North Carolina, USA). Since concentrations for IL-6 and IL-8 varied between runs, data were normalized to either unstrained control (Figure 1C) or CSDS strain profile (Figure 2H-I) for each paired flex run.
Human Cytokine Membrane Array
Conditioned media was analyzed using the Proteome Profiler Human Cytokine Array (R&D Systems) as directed by the manufacturer. The arrays were developed using WesternBright Sirius reagent (Advansta, San Jose, California, USA) on a C-Digit scanner (LI-COR, Lincoln, Nebraska, USA) and signal densities were determined using Image Studio software package (LI-COR). Data were normalized to CSDS strain profile for each paired flex run.
Cytometric Bead Array
Conditioned media was analyzed with the Human Pro-Inflammatory Cytokine Cytometric Bead Array kit (BD Biosciences, Franklin Lakes, New Jersey, USA) as directed by the manufacturer using a Accuri C6 Flow Cytometer (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA). Since concentrations for IL-6 and IL-8 varied between runs, data were normalized to CSDS strain profile for each paired flex run.
Statistical analyses were performed using GraphPad Prism 5 as described in each respective figure legend or in the text. A p-value of < 0.05 was considered significant.