Cyclic Stretch of Trabecular Meshwork Cells

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)).