M. Pabst, J. S. Bondili, J. Stadlmann, L. Mach and F. Altmann, Anal. Chem., 2007, 79, 5051–5057 CrossRef CAS PubMed. M. Wuhrer, C. A. Koeleman, C. H. Hokke and A. M. Deelder, Rapid Commun. Mass Spectrom., 2006, 20, 1747–1754 CrossRef CAS PubMed. Matsui TS, Kaunas R, Kanzaki M, Sato M, Deguchi S. Non-muscle myosin II induces disassembly of actin stress fibres independently of myosin light chain dephosphorylation. Interface Focus. 2011;1: 754–766. pmid:23050080 Farge E. Mechanical induction of Twist in the drosophila foregut/stomodeal primordium. Curr Biol. 2003;13: 1365–1377. pmid:12932320

Authors MN and TY are employees of Hemanext Inc. Authors TN and AD are founders of Omix Technologies Inc. and Altis Biosciences LLC. AD is an advisory board member of Hemanext Inc. and Forma Therapeutics Inc. PG is a statistical consultant of Hemanext Inc. Publisher’s note Gilbert JA, Weinhold PS, Banes AJ, Link GW, Jones GL. Strain profiles for circular cell culture plates containing flexible surfaces employed to mechanically deform cells in vitro. J Biomech. 1994;27: 1169–1177. pmid:7929466 The current protocols for glycomics analysis of cells often require a large quantity of biological material (4–10 × 10 6 cells), 11,22,23 and therefore are of limited use to decipher the glycosylation of cells that are available in minor amounts. Upon considerable modifications, with this approach we were able to analyze N- and O-glycosylation derived from 5 × 10 5 cells. Our aim was to provide a high throughput workflow suitable for cell glycosylation profiling ensuring sufficient material for glycomics analysis assuming different glycosylation characteristics of different biological samples. Therefore, we have not attempted to test the lowest cell amount necessary for the analysis of glycans derived from NMuMG cell line, as this would be specific for this cell line. However, PGC-MS based glycomics workflows have recently undergone major improvements with respect to sensitivity. 24–26 Using post-column make-up flow (PCMF) for enhancing sensitivity has shown potential to allow glycomics analysis from minor amounts of biological material such as rare cell populations as well as patient derived materials. Corning® 96-well EIA/RIA Easy Wash™ Clear Flat Bottom Polystyrene Not Treated Microplate, 25 per Bag, without Lid, Nonsterile Cells sense mechanical stretch to regulate their morphology and function [ 1]. Cellular response to stretch has been demonstrated to play a pivotal role in developmental process [ 2, 3] as well as in health maintenance [ 4]. One example includes the behavior of endothelial cells that orients their morphological polarization into a direction perpendicular to the direction of stretch upon heartbeat-induced cyclic stretch [ 5– 8]. This endothelial cellular response to orient away from stretch has been implicated in suppression of atherosclerosis to circumvent prolonged activation of pro-inflammatory signals [ 5, 9, 10].Echeverri CJ, Perrimon N. High-throughput RNAi screening in cultured cells: a user’s guide. Nat Rev Genet. 2006;7: 373–384. pmid:16607398 Sherman SA, Phillips JK, Costa JT, Cho FS, Oungoulian SR, Finan JD. Stretch injury of human induced pluripotent stem cell derived neurons in a 96 well format. Sci Rep. 2016;6: 34097. pmid:27671211 Did you know that there is another 96 channel instrument from INTEGRA which offers more channels (24, 384), working positions (up to 3), and hands-off time? Corning® 96-well EIA/RIA Easy Wash™ Clear Flat Bottom Polystyrene High Bind Microplate, 25 per Bag, without Lid, Nonsterile Corning® 96-well EIA/RIA Clear Round Bottom Polystyrene High Bind Microplate, 25 per Bag, without Lid, Nonsterile

Naruse K, Sokabe M. Involvement of stretch-activated ion channels in Ca2+ mobilization to mechanical stretch in endothelial cells. Am J Physiol Physiol. 1993;264: C1037–C1044. pmid:8386448Fig. 2 Analysis of N-glycans and O-glycans derived from bovine fetuin standard. (A) Combined extracted ion chromatograms (EIC) of N-glycans released from bovine fetuin standard. Blue square: N-acetylglucosamine, green circle: mannose, yellow circle: galactose, red triangle: fucose, right pointing pink diamond: α2,6-linked N-acetylneuraminic acid, left pointing pink diamond: α2,3-linked N-acetylneuraminic acid, H: hexose, N: N-acetylhexosamines, S: N-acetylneuraminic acid. (B) Inter- and intraday repeatability of the fetuin N-glycan analysis based on relative quantification of top 13 most abundant N-glycans. Inter- and intraday repeatability of the fetuin N-glycan analysis showed two median coefficients of variation (CV) of 7.6% and 8.0% within three technical replicates form the same plate, and a median CV of 9.8% within six technical replicates distributed into two plates over 1 month (displayed as mean relative abundance plus standard deviation; CVs of each glycans were listed on the top of the bar; intraday n = 3, interday n = 6, independent two different plates over 1 month). More details displayed in Table S1, ESI. † (C) Combined EICs of 5 O-glycans released from bovine fetuin standard, in which the top three most abundant O-glycans account for 98% of the relative abundance. Blue square: N-acetylglucosamine, yellow circle: galactose, pink diamond: N-acetylneuraminic acid, H: hexose, N: N-acetylhexosamines, S: N-acetylneuraminic acid. (D) Inter- and intraday repeatability of the top 3 most abundant O-glycans released from fetuin after removing N-glycans. (displayed as mean relative abundance plus standard deviation; CVs of each glycans were listed on the top of the bar; intraday n = 3, interday n = 6, derived from two different plates performed over 1 month). More details displayed in Table S2, ESI. † We observed that cells cultured in the elastic plate exhibited typical response to cyclic stretch to orient away from the direction of stretch ( Fig 6A). The cellular response to stretch is known to be repeatable because cells constantly sense changes in their mechanical environment and thus return to their original morphology with no preference in orientation upon static culture. This reversible cellular response was observed for over two days, demonstrating the capability of our device allowing for sufficiently long-term cell cultures ( Fig 6B). The present test also confirmed that the bottom of the PDMS membrane was attached to the plate body without leakage of culture media even during the whole process of the stretching.

Corning® 96-well Clear Flat Bottom Polystyrene TC-treated Microplate, Individually Wrapped, with Low Evaporation Lid, Sterile Previous multi-well plates with an elastic membrane attached to the bottom of the rigid plate body (i.e., a 96 well plate [ 28] or a 6 well plate [ 22– 25, 38]) allow for loading of radial (or biaxial) stretch with upward displacement of an underneath rigid post array or vacuuming. On the other hand, realizing uniaxial stretch under multi-well formats is technically difficult, while uniaxial stretch is widely present in tissues throughout the body including brain tissues [ 39] and thus important to study the cellular response. We mounted in our setup four guide rods to minimize the lateral shrink that impairs the application of uniaxial stretch, although they could not partly suppress the inevitable Poisson’s effect. The guide rods worked relatively well near the shorter edges of the plate, and consequently the compressive strains were relatively low there ( Fig 4D). In addition, some variations, larger in magnitude compared to the theoretical values from FEM ( Fig 3), appeared in all directions probably because of manufacturing defects ( Fig 4). We initially designed and fabricated a similar elastic plate with its well’s shape being square in top view because the strain distribution within individual wells is ideally further uniform compared to that within circular-shaped wells. However, we found in preliminary experiments that manufacturing defects were formed more obviously in the case of the square-well plate; thus, we decided to use more reliable circular wells in the present study. Another reason for the difference between the theoretical and experimental results includes the too restricted boundary conditions of the FEM model. The presence of the variations in applied strain level must be considered as a limiting factor; nevertheless, our device will be useful to enable high-throughput screening of molecules that change the activity depending on the presence or absence of uniaxial stretch. Corning® 96-well Clear Flat Bottom Polystyrene TC-treated Microplates, 10 per bag, with Lid, Sterile 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. Voyvodic PL, Min D, Baker AB. A multichannel dampened flow system for studies on shear stress-mediated mechanotransduction. Lab Chip. 2012;12: 3322–3330. pmid:22836694Corning® 96-well Clear Flat Bottom Polystyrene Not Treated Microplate, 20 per Bag, with Lid, Sterile Hahn C, Schwartz MA. Mechanotransduction in vascular physiology and atherogenesis. Nat Rev Mol Cell Biol. 2009;10: 53–62. pmid:19197332 Corning® 96-well EIA/RIA Clear Flat Bottom Polystyrene Not Treated Microplate, Individually Wrapped, without Lid, Nonsterile After narrowing down the candidate ASs to a more manageable number, they can be further investigated through more detailed studies including measurements of RBC deformability and P50, as well as more detailed metabolomics/lipidomics investigations using isotopes to elucidate the mechanism of action. The final candidate(s) can then proceed to a full-unit storage study in the final storage configuration, followed by a clinical study to determine post-transfusion recovery of stored autologous RBCs. Recently, an accompanying paper from Nemkov et al. has shown that the overall metabolic profile generated by this platform is comparable to that of RBCs stored in a standard storage bag, and successfully demonstrated the platform’s feasibility by applying it to examine the effect of spiking five different compounds into the AS-3 additive ( Nemkov et al., 2022). Spruell C, Baker AB. Analysis of a high-throughput cone-and-plate apparatus for the application of defined spatiotemporal flow to cultured cells. Biotechnol Bioeng. 2013;110: 1782–1793. pmid:23280552