Braakhuis HM, Park MV, Gosens I, De Jong WH, Cassee FR. How cells can control their size by pumping ions. 100 µL of GF suspension was added to 2 mL of DMEM to obtain a final concentration of 50 µg/mL. 2010;7:22. https://doi.org/10.1186/1743-8977-7-22. St?phane Collin. Carbon nanotubes hoax scientists in viability assays. It demonstrates the higher capability for planar CBNs to sequester proteins compared to cylindrical shaped CBNs. CAS  The population column refers to the gated group of cells measured to identify calcein (live) and PI (dead) populations. The results demonstrated that MWCNTs and GF induced cytotoxicity in fibroblast at different timescales. By using this website, you agree to our It is dependent upon temperature and pressure however. Nanomedicine. statement and Other reports have indicated that the similarity in shape and dimensions of MWCNTs with the actin stress fibers, once in the cell, could emulate the fibers, resulting in reinforcement of the cytoskeleton [31]; this effect could explain the results observed for NIH3T3 fibroblasts exposed to MWCNTs for 2 h. By contrast, a planar shape, such as that of the GFs, results in cellular membrane deformation associated with enhanced protein adsorption, a lower Young’s modulus and higher ROS production compared to those associated with exposure to MWCNTs, despite their lower SSA. The relative trigger force used was 2 nN to minimize the cell deformation and the damage to the cells. https://doi.org/10.1016/j.bpj.2013.12.037. Cells exposed to GFs did not show NMs in their cytoplasm, Fig. 1c; the GF remained outside the cells and appeared in some cases to adhere to the cellular membrane. Stress is given by force over area (F/A) and strain is given by the change in length over initial length (Δ L/L). https://doi.org/10.1002/lapl.201010087. 2012;33:4013–21. We also acknowledge Dr. John Gonzalez for his cooperation using the flow cytometer at the Faculty of Medicine, Universidad de Los Andes. Today's classroom version of the same experiment is typically performed using a laser beam as the source. Explicitly state where your pillow is behaving elastically? 2017;43:153–60. https://doi.org/10.1016/j.jtemb.2017.01.005. Pastrana, H.F., Cartagena-Rivera, A.X., Raman, A. et al. Correspondence to Lum H, Roebuck KA. Nevertheless, after 24 h of exposure to MWCNT the Young’s modulus dramatically decrease by 13.6% compared with 2 h results. Pflugers Arch. Part Fibre Toxicol. Therefore, cytoskeleton’s structures disruptions can be associated with cells metabolic stress. Then, cells were incubated for 2 and 24 h at 37 °C under 5% CO2 atmosphere. There is more protein adsorption, ten times, in GF regarding their SSA. J Nanoparticle Res. The NMs properties can also interfere with the reagents of the biochemical and optical assays leading to skewed interpretations and ambiguous results related to the NMs toxicity. https://doi.org/10.1039/C2IB20117H. Risk Anal. After 24 h, cells exposed to planar-shaped GFs produced twice as many ROS and exhibited a twofold decrease in Young’s modulus in contrast to cells exposed to cylindrical-shaped MWCNTs, even though that the specific surface area (SSA) of MWCNTs is double than the GFs SSA. Then the graph can be analysed to determine E. There are links from the PDF directly to the relevant part of the data collection in the video for anyone using the worksheet electronically. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This incongruency suggests the need for alternative techniques, to validate the results by a different mechanism not affected by biochemical interactions, such as cellular stiffness. Dulínska-Molak I, Mao H, Kawazoe N, Chen G, Duli I, Mao H, et al. Alba Ávila. Among the NMs properties, SSA has been widely accepted as the dominant toxicity predictor, since a greater SSA is associated with higher reactivity with cellular structures, in many cases due to a major ROS production [41]. https://doi.org/10.1371/journal.pone.0009445. Article  The LEAP images are presented for cells exposed to MWCNT and GF after 12 and 24 h measuring ROS production by DHE dye and live cells by Calcein dye. The excess of ROS results in additional glutathione residues to the actin monomers during the polymerization process of F-actin [19]. J Nanobiotechnol 17, 32 (2019). Finally, the stained cells were measured using a flow cytometer (FACS Canto II, BD Bioscience, San Jose, CA, USA; located at Universidad de Los Andes), which counted to 10,000 events for 30 s. Each group was seeded and tested three times, see Additional file 1. A review of atomic force microscopy applied to cell interactions with membranes. Bai H, Chen P, Fang H, Lin L, Tang GQ, Mu GG, et al. Young’s Modulus(Experimental) : 8465.746 Ksi 2. To satisfy this assumption, data were analyzed up to indentations of 20%-pixel cell height. Injury. The cells exposed to MWCNT shows almost two times the number of mitochondria and some vesicles with MWCNT inside, Fig. 1b. The reason is that the CBNs have several analytical obstacles, such as optical and chemical interference with current toxicity assay reagents, among them: adsorption of assay molecules by nanomaterials, increased reactivity of reagents, and shading effects of absorbance methods [2,3,4,5]. The samples were then centrifuged at 12,000 rpm for 10 min. We selected NIH3T3 fibroblasts since phagocytosis is not an active process in these cells [50]. Biophys J. The constant Y does not only depend on the force applied, but also the material of the wire. YOUNG’S MODULUS PRACTICAL OBJECTIVE • To determine the Young’s Modulus of a steel wire using a graphical method. Fluorescent staining of NIH3T3’s cytoskeleton: a control cell; F-actin (green) is highly organized with stress fibers extending continuously along the cells. Part Fibre Toxicol. 2 Apparatus required Graphene oxide flakes (XG Sciences Inc, Lansing, MI, USA) were suspended in 1× PBS at 1 mg/mL. Applying both conventional biochemical approach and cells mechanics, we were able to detect differences in the actin networks induced by MWCNT inside the cells and GF outside the cell’s membrane. Altogether, the changes in mechanical properties and the length of F-actin fibers confirmed that disruption of the F-actin cytoskeleton is a major consequence of cellular toxicity. Method for quantitative measurements of the elastic modulus of biological cells in AFM indentation experiments. J Mater Chem B. Cells were then seeded on a culture dish (Fluorodish, 50 mm, WPI, Sarasota, FL, USA) pre-coated with 0.1% gelatine in water (STEMCELL Technologies, Vancouver, British Columbia, Canada) at a concentration of 1.2 × 105 cells/mL. Simultaneous measurement of lateral and normal forces with an optical-beam-deflection atomic force microscope. Abbott LC, Maynard AD. The cells were exposed to 50 µg/mL of CBNs for 24 h. Then were fixed and prepared for TEM imaging. Metabolic regulation of stem cell function. 2007;45:1425–32. Significant at p < 0.05, n: 1000 points per group, t-test. The ability to adsorb proteins by planar CBNs has been extensively reported [36]. It is a measure of the stiffness of a material that is independent of the particular sample of a substance. Integrating physiological regulation with stem cell and tissue homeostasis. J Trace Elem Med Biol. https://doi.org/10.1016/j.carbon.2007.03.033. Young’s modulus, also known as the tensile modulus, elastic modulus or traction modulus (measured in Pa, which is a pressure unit(N.m^-2; 10MPa is equivalent to 1Kg force per square millimeter) is a mechanical property of linear elastic materials. https://doi.org/10.1126/science.1116995. We focused the characterization of MWCNT and GF on the main physical–chemical properties related to cells’ toxicity: size/size distribution, shape, surface area, composition, impurities, and surface charge [40]. Chai Hao. Young's Modulus Experiment Lab report for Youngs Modulus Experiment. Our study is focused on the cellular mechanical response, which mainly depends on cytoskeletal integrity [17]. Figure 1: Young’s Modulus Comparison. The lack of F-actin filaments results in lower Young’s modulus values [20,21,22,23,24]. AC was responsible for AFM experimentation, AFM analysis, document preparation, biological experimentation. https://doi.org/10.1111/ropr.12129. Also, we evidenced that after 2 h of exposure to GFs the cells’ volume decrease. Samples were not sonicated to simulate a direct biological contact under normal NMs exposure in the environment. The more detailed results are provided below. Samples were not sonicated as well as MWCNT. Oxidant stress and endothelial cell dysfunction. However, no analysis of cells stiffness at multiple points of the cells were done to map the cell localized heterogeneity, as wells as the assessment of the extracellular effects and dimensionality of the nanomaterials were done. In summary, our results suggest that toxicity assessments need to consider additional techniques apart from the traditional, here demonstrated with the integration of the single the cells’ mechanical properties. 2016;110:2528–39. 2015;10:2423–50. Generally, dynamic Young’s modulus, dynamic shear modulus and Poisson’s ratio are considered to be the primary results of this testing standard. Raman spectroscopy was used to structurally characterize both nanomaterials (MWCNTs and GFs) in powder form. 2013;110:16027–32. Questions 1. The fiber length was measured by image processing on ImageJ skeleton algorithm. Previous works have concentrated on studying the toxicity effects based on the SSA [41]. 2013;5:66–73. However, MWCNTs seems to exert more intracellular effects interfering with cellular structures and breaking lysosomal vesicles increasing the ROS production [26]. 2 Results. https://doi.org/10.1152/ajpcell.2001.280.4.C719. Hirsch C, Roesslein M, Krug HF, Wick P. Nanomaterial cell interactions: are current in vitro tests reliable? Is your pillow an elastic material? https://doi.org/10.1016/j.toxlet.2006.11.001. Lawen A, Lane DJR. The yellow arrow indicates a GF. Subsequently, calcein-AM green dye (Life Technologies, Carlsbad, CA, USA) and propidium iodine (PI) (Life Technologies, Carlsbad, CA, USA) were added to stain the cells for 30 min in the dark at room temperature. We used the standard force–volume AFM mode to generate topographic, and elasticity map sought to determine the cell’s stiffness (Young’s modulus) of untreated and CBN-treated (MWCNT and GF) fibroblasts after 2 and 24 h of exposure, to 50 µg/mL concentration, Fig. 5. Young’s modulus differences between untreated cells and cells exposed to MWCNTs and GFs. https://doi.org/10.1042/BJ20100208. We found that the greater SSA of MWCNT were not a good predictor of cells toxicity by CBNs as their shape and dimensionality. The Young Modulus is also known as Young's Modulus or the elastic modulus or tensile modulus.. Pulskamp K, Diabaté S, Krug HF. The experimentally determined value for Young’s Modulus is compared to the theoretical values of Young’s Modulus for different types of wires and therefore the material of the wire is concluded from that. Methods. https://doi.org/10.1089/ars.2011.4271. 3. Thus, we observed that the shape of CBN strongly affects the cellular cytotoxicity than their SSA. Burgess RJ, Agathocleous M, Morrison SJ. Cells were maintained at 37 °C during the AFM experiments using a Petri dish holder and a heater on the MFP-3D scanner X–Y stage (Asylum Research, Santa Barbara, CA, USA). https://doi.org/10.1002/jcp.1041190110. These results contrast with the conventional live/dead assay where we obtained viabilities greater than 80% after 24 h; while the elasticity dramatically decreased suggesting a fast-metabolic stress generation. PLoS Comput Biol. Secondary to the glutathionylation, the F-actin cytoskeleton no longer possesses straight filaments across the cytoplasm, but instead an aggregation of actin monomers. Cells exposed to GFs resulted in shorter actin fibers after 24 h than the fibers in cells exposed to MWCNT. Young’s Modulus or Elastic Modulus or Tensile Modulus, is the measurement of mechanical properties of linear elastic solids like rods, wires, etc. J Funct Biomater. https://doi.org/10.1111/j.1539-6924.2010.01446.x. Science. Discher DE, Janmey P, Wang Y-L. Tissue cells feel and respond to the stiffness of their substrate. https://doi.org/10.1021/jp208967t. The cells are exposed to CBN, the presence of these materials in the media induce ROS production, protein adsorption, and F-actin polymerization disruption. Protein concentration in the supernatant after exposure to CBNs for 24 h at a concentration of 10 µg/mL and 50 µg/mL. Rev Policy Res. Therefore, it is possible to have stressed cell also labeled as viable by calcein marker. Illner D, Scherthan H. Ionizing irradiation-induced radical stress stalls live meiotic chromosome movements by altering the actin cytoskeleton. https://doi.org/10.1016/j.immuni.2012.08.017. PubMed Central  Subramanian V, Semenzin E, Hristozov D, Zabeo A, Malsch I, McAlea E, et al. https://doi.org/10.1186/s12951-019-0460-8, DOI: https://doi.org/10.1186/s12951-019-0460-8. Our experimental protein absorption results demonstrated higher adsorption rates for GFs than for MWCNTs, which led to a significant decrease of the availability of proteins for cell homoeostasis, increasing the ROS production after 24 h of exposure to 32% for cells exposed to GF and 17% for cells exposed to MWCNTs. For example, the fibroblasts’ cytoskeleton is rich in actin fibers. 2014. https://doi.org/10.2788/406937. 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