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Recent Advances in Imaging and Analysis of Cellular Dynamics in Real Time
Published in Jyoti Ranjan Rout, Rout George Kerry, Abinash Dutta, Biotechnological Advances for Microbiology, Molecular Biology, and Nanotechnology, 2022
Chandra Bhan, Pankaj Dipankar, Shiba Prasad Dash, Papiya Chakraborty, Nibedita Dalpati, Pranita P. Sarangi
For visualization of biological properties of a living cell and the detailed dynamics of biological and physiological processes in real time, it is essential to provide the cells an optimal physiological environment during a live-cell imaging experiment. Stringent control of the culture environment is an important factor in acquiring information during live-cell imaging and the accuracy of data collected depends largely on the maintenance of cells on the microscope stage. Health of a cell is comparatively less critical during short-term experiments, while for long-term experiments it is imperative to watch out all the possible factors such as (1) pH, (2) oxygenation, (3) temperature, and (4) osmolarity, which could alter metabolic function of the specimen (Daniels, 2012; Cole, 2014). pH: Most of the mammalian cell line show optimal growth in a pH range of 7.2–7.4 and maintenance of CO2 concentration in the chamber is crucial to regulate the physiological pH during the entire imaging process. Cells usually have an optimal growth condition when supplemented with 10% fetal bovine serum and 5% of CO2 concentration. HEPES buffer at a concentration of 10–20 mM is often used as a buffering agent due to its cost-effectiveness for a short period of experiments. But, for long-term experiments, which last usually more than 10–12 h, HEPES buffer is not recommended (Daniels, 2012).Oxygenation: Oxygenation is not a concern for short-term live-cell imaging techniques, but it is a critical factor for cell health during longer experiments. To manage the oxygenation during long-term experiments, the media could be changed frequently or a larger quantity of media can be used at the beginning of the experiment. In addition, imaging chambers could be connected with inlets for required gases such as oxygen and carbon dioxide.Temperature: Temperature serves as the most critical factor that decides the outcome of the experiment. The cellular physiology will be disturbed even with a smaller variation in temperature parameter. To maintain the optimum temperature, either a small stage-top incubator can be used to maintain sample temperature, or large incubators could be used to heat the entire microscope. The objective can act as a heat sink during live-cell imaging experiment. Hence, objective heaters are designed to overcome this situation. Metal foil blanket with Velcro anchor, copper tubing water jacket, and proportionally controlled closed-loop heaters are some of the designs used to thermally control the objectives (Daniels, 2012).Osmolarity: Osmolarity is maintained in a range of 260–320 mOsm/ kg during live-cell imaging, which is achieved by averting the evaporation of culture media by using properly sealed culture chambers. Alternatively, a humidified environment can be maintained to achieve the required osmolarity.
Stepwise Recovery of Molybdenum, Vanadium, and Tungsten with Amino-Type “Trident” Molecule by Stripping
Published in Solvent Extraction and Ion Exchange, 2021
Keisuke Ohto, Hiroaki Furugou, Shintaro Morisada, Hidetaka Kawakita, Ken-ichi Isono, Katsutoshi Inoue
Metal ions were extracted using the amino compounds using a similar method to that used in a previous study.[44] The organic phase was prepared by dissolving 3A in 1-octanol or chloroform to give the desired concentration (5.0 mM). The aqueous phase was prepared by dissolving the appropriate amount of the desired metal nitrate salt to give a concentration of 0.10 mM of the mononucleated species in water containing 0.10 M nitric acid and 0.10 M 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) or, if appropriate, 0.10 M NaOH. The HEPES pKa is 7.55. The HEPES was used to buffer the aqueous solution even though the experiments were performed at relatively acidic pH values. The aqueous solutions were mixed as necessary to adjust the pH. A mixture of 5.0 cm3 of both the organic and aqueous phases was prepared and shaken for 24 h at 30°C. The phases were allowed to separate, and then the pH and metal concentrations were measured using a pH meter and an ICPS-8100 inductively coupled plasma atomic emission spectrophotometer (Shimadzu, Kyoto, Japan), respectively. The percentage extracted (%Extraction) was calculated using Equation (2),
Tunable nonenzymatic degradability of N-substituted polyaspartamide main chain by amine protonation and alkyl spacer length in side chains for enhanced messenger RNA transfection efficiency
Published in Science and Technology of Advanced Materials, 2019
Mitsuru Naito, Yuta Otsu, Rimpei Kamegawa, Kotaro Hayashi, Satoshi Uchida, Hyun Jin Kim, Kanjiro Miyata
β-Benzyl-l-aspartate N-carboxyanhydride (BLA-NCA) was purchased from Chuo Kaseihin Co. Inc. (Tokyo, Japan). n-Butylamine and 1,3-diaminopropane (DAP) were purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). N-Methyl-2-pyrrolidone (NMP), benzene, N,N-dimethylformamide (DMF), dichloromethane (CH2Cl2), thiourea, lithium bromide (LiBr), dimethyl sulfoxide-d6 (containing 0.05 vol% tetramethylsilane), ethylenediamine (EDA), 1,4-diaminobutane (DAB), sodium chloride (NaCl), acetic acid, deuterium oxide, sodium 3-(trimethylsilyl) propionate-2,2,3,3-d4, glycine, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate 12-water, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, and Dulbecco’s phosphate-buffered saline (D-PBS) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) was purchased from Dojindo Laboratories (Kumamoto, Japan). Trypsin-ethylenediamine tetraacetate and penicillin-streptomycin (P/S) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). EDA, DAP, DAB, NMP, and DMF were distilled with calcium hydride (CaH2) under reduced pressure. CH2Cl2 was distilled with CaH2 under normal pressure. A human hepatoma cell line (Huh-7) was obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). Huh-7 cells were cultured in DMEM with 10% FBS (v/v) containing 1% P/S and incubated in 5% CO2 at 37 °C.
Substitution behavior of square-planar and square-pyramidal Cu(II) complexes with bio-relevant nucleophiles
Published in Journal of Coordination Chemistry, 2018
Enisa Selimović, Andrei V. Komolkin, Andrei V. Egorov, Tanja Soldatović
In order to determine the metal–ligand stoichiometry, a series of 10-mL solutions was prepared in which the concentration of [CuCl2(en)] or [CuCl2(terpy)] complexes was held constant (0.0001 M) while the concentration of chloride was varied in different molar ratios ([Cl−]/[CuCl2(en)] = 1, 2, 3, 5, 10, 15, 20, 30, 40, and 50) [25, 26]. Hepes (0.025 M) was used as a buffer at pH 7.4. The absorbance of each solution was measured over the wavelength range 200–900 nm. An increase of the absorbance around the maximum at 220 nm and a decrease at the minimum at 300 nm, with an isosbestic point at 340 nm, has been noticed. The isobestic point at 340 nm indicates the presence of different complex species and physical changes in the system. The absorbance at 300 nm was plotted vs. the molar ratio of the reactants (Figure 2). Assuming the formed complex absorbs more than the initial reactants, this plot produces an increasing absorbance up to the combining ratio. At this point, further addition of chloride produces less increase in absorbance. Thus, a break in the slope of the curve occurs at the mole ratio corresponding to the combining ratio of the chloride/complex.