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Functional Study of Lysosomal Nutrient Transporters
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Xavier Leray, Corinne Sagné, Bruno Gasnier
We provide here an example of amino acid counter-transport protocol used to characterize the lysosomal transporter for glutamine and asparagine SNAT7 (Verdon et al., 2017). Quantities are given to test up to four experimental conditions measured in triplicate at a single time point. If more conditions must be tested it is preferable to run separate experiments rather than scaling up the number of cells because lysosomal integrity decreases with lengthier cell fractionation. Alternately, cell homogenization can be done with a Parr cell disruption bomb or a BioNeb nebulizer to scale up the protocol without lengthening the homogenization step.
Dialyzable and Nondialyzable Transfer Factor
Published in Edward P. Cohen, A. Arthur Gottlieb, Immune RNA, 2020
The procedures used to prepare TFd and TFnd are usually those originally formulated by Lawrence5 using peripheral blood leukocytes. Peripheral blood is collected into any one of a number of anticoagulants, heparin, ACD solution, or ethyl-enediaminetetracetate. The leukocytes are separated from the red blood cells and plasma. Spleen and lymph nodes are usually processed to produce single cell suspensions. The cells are washed and then disrupted. It is important that the cells be processed quickly. Many investigators have observed that lysates prepared from cells allowed to stand for several hours before disruption do not have any TF activity. Washed centrifuged cell pellets may be disrupted by freezing at -70°C and thawing at 37°C. Usually, 6 to 10 cycles are required to completely lyse the cell pellet. Alternate methods of cell disruption include sonication or N2 decompression. The lysates may be treated with DNAse at this point.5 DNAse treatment decreases the viscosity of the lysate, but it is not essential for the production of TFd andTFnd.7,8
Seeing with Sound: Diagnostic Ultrasound Imaging
Published in Suzanne Amador Kane, Boris A. Gelman, Introduction to Physics in Modern Medicine, 2020
Suzanne Amador Kane, Boris A. Gelman
A phenomenon called cavitation is mainly responsible for mechanical cell disruption. You may have heard that water boils at a lower temperature at higher altitudes, due to the lower pressures encountered there. Similarly, in cavitation, the rarefactions due to a very intense sound wave can create pressures so low that bubbles of water vapor can form even at body temperature. Cavitation due to exposure to very high-intensity ultrasound can result in small bubbles forming and collapsing so as to act like tiny explosions that can disrupt cell membranes and internal structures. In fact, ultrasound tools used for surgery produce damage by this very means. Both of these effects have been carefully studied in the laboratory to establish at what intensity levels significant temperature rises and signs of cavitation occur so diagnostic imaging can take place at far lower intensities.
Evaluation of cytotoxicity, analysis of metals and cumulative risk assessment in microalgae
Published in Toxicology Mechanisms and Methods, 2023
Mercedes Taroncher, Yelko Rodríguez-Carrasco, Francisco J. Barba, María José Ruiz
There is a growing demand for renewable high value carbohydrates, proteins, lipids, polyphenols and pigments as an alternative for traditional agricultural crops. Microalgae are one of the most promising sources for producing these biomolecules (Postma et al. 2016) which can be used as supplementary food or feed (Postma et al. 2017). In this sense, the European Food Safety Authority (EFSA) and the Spanish Agency for Consumer Affairs, Food Safety and Nutrition (AECOSAN) within the scope of Regulation (EC) No 258/1997 on novel foods and novel food ingredients have authorized to market dried microalgae Tetraselmis chuii in food supplements in the European Union. And the microalgae Phaeodactylum tricornutum and its derived oils, also considered a novel food, are permitted for consumption as food supplements by the general population in the European Union (EU). These microalgae consumption under the conditions proposed by the company reveals that no adverse effects on human health are produced (Calderon Pascual et al. 2017). For instance, natural carotenoids are more easily absorbed by animals than those obtained via chemical, that is because there is an increase demand for carotenoids obtained from natural sources such as microalgae (Martínez et al. 2018). Such products are normally located intracellular, in the cytoplasm, in internal organelles or bound to cell membranes, so the cells need to be disintegrated before extraction. Thus, the evaluation of the cell disruption (DR) yield is therefore of great importance.
B-Raf inhibitor vemurafenib counteracts sulfur mustard-induced epidermal impairment through MAPK/ERK signaling
Published in Drug and Chemical Toxicology, 2023
Zhiyong Xiao, Feng Liu, Junping Cheng, Ying Wang, Wenxia Zhou, Yongxiang Zhang
The viability of HaCaT cells, as measured by the CCK-8 assay, reduced after 6 h of SM challenge at 500 μM concentration (Figure 1(a)), and the viability of HaCaT cells decreased by 25% and 70% after 24 h of treatment with 100 and 500 μM SM, respectively (Figure 1(b)). Vemurafenib increased cell survival rates after 24 h of exposure to 500 μM SM (Figure 1(b)). Cellular morphology was also directly investigated using the dynamic monitoring platform IncuCyte ZOOM™. As shown in Video A, HaCaT cells underwent a dramatic change in shape, and cell disruption levels gradually increased with time after exposure to 500 μM SM. Vemurafenib attenuated the SM-induced shrinkage of HaCaT cells (Figure 1(c), Figure S2). The intracellular ATP level diminished by 27% after 6 h of exposure to 500 μM SM, and the ATP level seemed to be slightly restored after the addition of vemurafenib (Figure 1(d)). After 24 h of SM exposure, the ATP level increased from 20% to 35% with the addition of vemurafenib (Figure 1(e)).
Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles
Published in Journal of Extracellular Vesicles, 2020
Javier Mariscal, Tatyana Vagner, Minhyung Kim, Bo Zhou, Andrew Chin, Mandana Zandian, Michael R. Freeman, Sungyong You, Andries Zijlstra, Wei Yang, Dolores Di Vizio
Whole cell lysate (WCL) and membrane preparations (M) were obtained upon 24-hour serum starvation and collection of conditioned cell media. Cell monolayers were scraped and washed in chilled PBS (×3). For WCL, cells were directly lysed in DTT-free 4% SDS/Tris-HCl lysis buffer [25]. For M preparations, cells were gently scraped, washed in PBS (×3) and resuspended in filtered PBS containing 1% protease inhibitors (cOmplete Mini Protease Inhibitor Cocktail, Roche). Cell suspensions were immediately subjected to 20 cycles of sonication (5 sec) in ice to induce cell disruption. Membrane suspensions were then cleared of intact cells at 500 g, pelleted at 16,000 g (20 min, 4°C), washed in PBS (×3) and resuspended in 4% SDS/Tris-HCl lysis buffer. All protein lysates were stored at −80°C until use.