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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. Table 1A shows the ATP values for the N and for H RBCs throughout the 6-week storage accompanied by descriptive statistics and the results of analysis by a mixed model analysis of variance ( Table 3). Higher ATP levels were observed for samples stored hypoxically, as reported previously ( Yoshida and Shevkoplyas, 2010; Dumont et al., 2016; Yoshida et al., 2022) with an overall estimated difference = 0.48µmol/gHb, p≤ 0.0001. The three observed ATP profiles of the samples stored in the 96-well plate with PVC strips were similar to the values and trends seen in samples stored in PVC bags, both in N and H storage conditions over the 6-week study period. There was no significant estimated difference within the H samples regardless of bag or plate storage, number of PVC strips, or time point throughout the study. Within the N samples, a small but significant estimated difference (0.66µmol/gHb p< 0.004) in ATP was predicted by the model for the bag storage compared to the plate storage with no PVC ( Table 3). Other parameters Corning® 96-well Clear Round Bottom Polystyrene Not Treated Microplate, 20 per Bag, with Lid, Sterile Now, let’s look at how the colour wheel impacts your chosen shade. In the Wella portfolio, each hue is numbered to show the depth, followed by a digit that denotes the major tone then, often, a minor tone. As seen on the left, colours with a 6 after the dash will have a violet tonal direction. As violet is the complementary colour for yellow, you can decide whether this 6 should be the major or the minor, depending on how yellow strands are. The proposed platform’s equivalence is further demonstrated by additional biochemical parameters such as pH, lactate and K + ( Figure 3). As expected, due to H processing and subsequent storage, there were higher lactate levels observed for H samples when compared to N samples ( Figure 3A), reflecting an enhanced glycolytic flux from H storage ( Dumont et al., 2016; Yoshida and Shevkoplyas, 2010; D'Alessandro et al., 2020). Lower pCO 2 and higher initial pH observed for H samples were mainly due to CO 2 depletion during the O 2 reduction process ( Figure 3B) and the observed pH cross-over mid-storage ( Figure 3B) can be ascribed to the increased rate of lactate accumulation during H storage ( Dumont et al., 2016). On the other hand, there was no noticeable difference in K+ between any of the samples, regardless of time point, strip number, storage method (plate vs. bag) or storage condition (H or N).
Corning® 96-well Half Area Clear Flat Bottom Polystyrene High Bind Microplate, 25 per Bag, without Lid, Nonsterile Corning® 96-well Half Area Clear Flat Bottom Polystyrene Not Treated Microplate, 25 per Bag, without Lid, Nonsterile Corning® 96-well Clear Round Bottom Polystyrene Not Treated Microplate, 25 per Bag, without Lid, Sterile
S. Holst, G. W. van Pelt, W. E. Mesker, R. A. Tollenaar, A. I. Belo, I. van Die, Y. Rombouts and M. Wuhrer, Methods Mol. Biol., 2017, 1503, 185–196 CrossRef CAS PubMed.
T. Nguyen-Khuong, A. Pralow, U. Reichl and E. Rapp, Glycoconjugate J., 2018, 35, 499–509 CrossRef CAS PubMed. Hirata H, Tatsumi H, Sokabe M. Mechanical forces facilitate actin polymerization at focal adhesions in a zyxin-dependent manner. J Cell Sci. 2008;121: 2795–2804. pmid:18682496 Deguchi S, Hotta J, Yokoyama S, Matsui TS. Viscoelastic and optical properties of four different PDMS polymers. J Micromech Microeng. 2015;25: 97002.Corning® 96-well EIA/RIA Clear Flat Bottom Polystyrene Not Treated Microplate, 25 per Bag, without Lid, Nonsterile Electronic supplementary information (ESI) available: Table S1: Relative quantification of N-glycans released from fetuin standard using a 96-well plate sample preparation method and PGC nano-LC-ESI-MS/MS. Table S2: Relative quantification of O-glycans released from fetuin standard using a 96-well plate sample preparation method and PGC nano-LC-ESI-MS/MS. Table S3: N-glycans released from NMuMG cell line using a 96-well plate sample preparation method and PGC nano-LC-ESI-MS/MS. Table S4: Relative quantification of N-glycans released from NMuMG cells using a 96-well plate sample preparation method and PGC nano-LC-ESI-MS/MS. Table S5: Relative quantification of O-glycans released from NMuMG cells using a 96-well plate sample preparation method and PGC nano-LC-ESI-MS/MS. Fig. S1: Elution pattern of N-glycans with and without phosphate on PGC. See DOI: 10.1039/c9mo00180h Both hemolysis and ATP values were analyzed separately using a mixed model analysis of variance, with a random effect of RBC pool, to estimate the effects of storage method and differences over time. In all analyses, the dependent variable was hemolysis or ATP at weeks 2, 4, and 6, and the independent fixed effect variables were baseline pool value, time point (week 2, 4, 6), process method (N or H), storage method (0, 1, or 2 PVC strips, or bag), interaction of time point and storage method, interaction of process method and storage method, interaction of process method and time point, and interaction of process method and storage method and time point. In either case Bonferroni multiple adjustment procedures were implemented. Results Red blood cell storage under N and H conditions: sO 2 and pCO 2 Deguchi S, Kudo S, Matsui TS, Huang W, Sato M. Piezoelectric actuator-based cell microstretch device with real-time imaging capability. AIP Adv. 2015;5: 67110. Putnam AJ, Cunningham JJ, Dennis RG, Linderman JJ, Mooney DJ. Microtubule assembly is regulated by externally applied strain in cultured smooth muscle cells. J Cell Sci. 1998;111: 3379–87. Available: http://www.ncbi.nlm.nih.gov/pubmed/9788879 pmid:9788879
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]. M. Pabst, J. S. Bondili, J. Stadlmann, L. Mach and F. Altmann, Anal. Chem., 2007, 79, 5051–5057 CrossRef CAS PubMed. E. D. Green, G. Adelt, J. U. Baenziger, S. Wilson and H. Van Halbeek, J. Biol. Chem., 1988, 263, 18253–18268 CAS. Its tiny size and portability allow the instrument to be easily moved for use anywhere in the lab, from the LAFC to small work spaces on any lab bench. The MINI 96 is also easy to use – simply turn it on and start pipetting.Once a promising AS is identified, this platform can then also be adopted to screening the ASs’ universal compatibility against a large number of individual RBC units using RBCs in segments.
I. Trbojevic-Akmacic, M. Vilaj and G. Lauc, Expert Rev. Proteomics, 2016, 13, 523–534 CrossRef CAS PubMed. H. Hinneburg, P. Korac, F. Schirmeister, S. Gasparov, P. H. Seeberger, V. Zoldos and D. Kolarich, Mol. Cell. Proteomics, 2017, 16, 524–536 CrossRef CAS PubMed. To improve the efficiency of the initial screening stage, we examined the use of deep 96-well plates for RBC storage in various ASs using hemolysis and ATP as the primary evaluation metrics. These parameters were chosen as they are easily adopted to a 96-well workflow and allow for a sufficiently comprehensive initial characterization of the novel ASs: ingredients incompatible with RBC storage are screened out by hemolysis, and gross metabolic effects are identified by ATP levels. Results: Final ATP and hemolysis values for the plate-stored RBCs were comparable to the typical values observed for 6-week storage of leukoreduced AS-3 RBCs in PVC bags under both N and H conditions. Hemolysis was below FDA and EU benchmarks of 1% and 0.8%, respectively, and excluding DEHP from plates during storage, resulted in an inconsequential increase when compared to bag samples.H. Hamouda, M. Kaup, M. Ullah, M. Berger, V. Sandig, R. Tauber and V. Blanchard, J. Proteome Res., 2014, 13, 6144–6151 CrossRef CAS PubMed. Another challenge in the field is the increasing need for high-throughput sample preparation for glycan analysis, due to a vast increase of sample numbers and complexity required in functional glycomics, systems biology, and clinical applications. Currently, there are several high-throughput approaches for N-glycan analysis (mainly glycoprotein), 9–11 while less methods are available for O-glycomics. 12 Therefore, there is a high demand for high-throughput, reproducible and robust analytical methods for integrated N- and O-glycomics. Scaling up the conventional glycomics analysis to higher-throughput approaches especially with respect to the sample preparation workflow is of great importance for ensuring sufficient sample size providing more reliable results. Fig. 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. † Chagnon-Lessard S, Jean-Ruel H, Godin M, Pelling AE. Cellular orientation is guided by strain gradients. Integr Biol. 2017;66: 409–422. pmid:28534911 Corning® 96-well EIA/RIA Clear Round Bottom Polystyrene Not Treated Microplate, 25 per Bag, without Lid, Nonsterile
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