Neumann B, Held M, Liebel U, Erfle H, Rogers P, Pepperkok R, et al. High-throughput RNAi screening by time-lapse imaging of live human cells. Nat Methods. 2006;3: 385–390. pmid:16628209

Corning® 96-well Clear Round Bottom Polystyrene Treated Microplate, 25 per Bag, without Lid, Nonsterile, Special ProcessEcheverri CJ, Perrimon N. High-throughput RNAi screening in cultured cells: a user’s guide. Nat Rev Genet. 2006;7: 373–384. pmid:16607398 Hahn C, Schwartz MA. Mechanotransduction in vascular physiology and atherogenesis. Nat Rev Mol Cell Biol. 2009;10: 53–62. pmid:19197332

To evaluate the actual strain of the plate in response to motions of the motors connected via several mechanical links, the entire plate was imaged during cyclic stretching ( Fig 2B). Quantification of the captured sequential images indicated that indeed the entire plate exhibited a desired sinusoidal 7% stretch pattern as consistent with the programed motions of the motors ( Fig 2C). Next, we evaluated strains at the individual well level by microscopy. First, we mapped the longitudinal and transverse strains of wells in the major and minor axes, respectively, by measuring the difference in the outermost edges ( Fig 4B and 4C). These quantitative data indicated that the longitudinal strains reached, upon the macroscopic 7% stretch exerted on the shorter plate edges, 11.6% ± 1.3% (mean ± SD) for “Columns A1–H1 and A12–H12”, 11.0% ± 1.2% for “Rows A1–A12 and H1–H12”, and 11.1% ± 0.8% for “Central wells” ( Fig 4E). Thus, we found that the longitudinal strains obtained in the experiments can be variable and exceed on average the theoretical value 10%, but the magnitude was almost comparable to the prediction by FEM. The transverse strains in the same condition were -0.7% ± 0.9% for “Columns A1–H1 and A12–H12”, -1.9% ± 0.9% for “Rows A1–A12 and H1–H12”, and -1.1% ± 0.6% for “Central wells” ( Fig 4D). Here, a statistically significant difference was present between central and peripheral wells ( Fig 4D) so that in the following local strain analyses we focused only on the central wells.Cellular response to stretch has been extensively studied, but conventional experimental systems are often constituted from single or only 6 wells partly because the majority of the previous studies focused on imaging of the dynamics of specific individual cells/molecules [ 13, 18, 32– 37] or examining changes in mRNA expression [ 22, 38] rather than performing an assay for molecular screening, the last of which generally requires huge numbers of screening trials. For example, a screening study was recently reported to reveal the Rho-GEFs responsible for the cyclic stretch-induced repolarization from 63 candidate molecules [ 6]. Here, they employed a stretch chamber with a single well of a 20 x 20 mm 2 cell culture area and a Rho-GEFs-targeted shRNA library, which we guess might took long time to complete and was costly to prepare large amounts of reagents. In this regard, our strategy of combining the stretch chamber and library directly at the small individual well levels can highly improve the screening throughput and cost. In addition to such candidate gene screenings, our system is useful as well in compound screening with various reagent libraries to suppress or rescue cellular responses altered by gene mutations. Corning® 96-well Clear V-Bottom Polystyrene Not Treated Microplate, Individually Wrapped, without Lid, Sterile

D. J. Harvey, M. Edgeworth, B. A. Krishna, C. Bonomelli, S. A. Allman, M. Crispin and J. H. Scrivens, Rapid Commun. Mass Spectrom., 2014, 28, 2008–2018 CrossRef CAS PubMed.Matsui TS, Ito K, Kaunas R, Sato M, Deguchi S. Actin stress fibers are at a tipping point between conventional shortening and rapid disassembly at physiological levels of MgATP. Biochem Biophys Res Commun. 2010;395: 301–306. pmid:20353757 Corning® 96-well Clear Flat Bottom Poly-D-Lysine Coated Microplate, 20per Bag, with Lid, Aseptically Manufactured We tested the plate storage under the regular normoxic (N) condition in ambient air, as well as under an oxygen and CO 2 controlled condition—‘anaerobic’, or hypoxic/hypocapnic (H) condition, as the latter has been shown to produce a variety of benefits including a reduction in oxidative stress and the reprograming of RBC metabolism in beneficial ways ( D'Alessandro et al., 2020) ( Yoshida and Shevkoplyas, 2010). As shown in Figure 2, the main characteristic difference between the two conditions is determined by oxygen content of the RBCs measured as hemoglobin oxygen saturation (sO 2), and pH due to pre-storage reduction of O 2 and pCO 2. A large and expected difference in the sO 2 was maintained between N and H samples throughout the study ( Figure 2A) as intended. Under N conditions a noticeable difference of sO 2 was observed between the bag and the plate samples ( Figure 2A), which can be attributed to the bag having a larger surface area exposed to ambient air and allowing for higher levels of oxygenation due to O 2 diffusion during storage ( Yoshida et al., 2022). Additionally, metallic foil seals were used for both types of plates, which eliminated O 2 ingress from the top. For the N plates, only the surface area on the bottom and sides was exposed to ambient air, and due to the relatively low O 2 permeability of the rigid polypropylene, average sO 2 remained stable except for the initial sO 2 increase which occurred during the filling of the wells in ambient air. There was also a small difference in sO 2 within the H plate samples, especially between the 0 and 2 strip samples ( Figure 2A). Further investigation is needed to explain this observation, although such a small difference in sO 2 is physiologically insignificant. The typical pCO 2 profile and difference in pCO 2 values between the N and H conditions observed in bag stored samples ( Dumont et al., 2016) were replicated in the plate platform ( Figure 2B). Higher pCO 2 levels observed for the normoxic bag sample were likely a result of higher flux through the oxidative pentose pathway triggered by higher oxygen levels.