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Glomerular Barrier Behaves as an Atomically Precise Bandpass Filter in a Sub-nanometre Regime *
Published in Valerio Voliani, Nanomaterials and Neoplasms, 2021
Du Bujie, Xingya Jiang, Anindita Das, Qinhan Zhou, Yu Mengxiao, Rongchao Jin, Jie Zheng
To test whether these AuNCs bind to serum protein or not, Au10-11 SG10-11, Au15SG13, Au18SG14 and Au25SG18 were incubated with either PBS or PBS supplemented with 10% (vol/vol) fetal bovine serum (FBS) at 37°C for 30 min. In addition, Au10-11SG10-11, Au18SG14 and Au25SG18 were incubated with PBS or PBS supplemented with 10% (vol/vol) FBS at 37°C for 24 h to test whether these AuNCs bind to serum protein after long-term (24 h) incubation with FBS. To identify the colourless protein band, FBS-incubated Au10-11SG10-11, Au15SG13, Au18SG14 and Au25SG10and pure FBS were stained by 10% (vol/vol) Coomassie Brilliant Blue 250 (CBB 250). All samples were analysed using 2% agarose gel electrophoresis (Mini-sub cell GT gel electrophoresis system from Bio-Rad). The amount of gold in each band was quantified using ICP-MS.
Prolactin Receptors in Normal Tissues and in Animal Models for Breast Cancer
Published in Nagasawa Hiroshi, Prolactin and Lesions in Breast, Uterus, and Prostate, 2020
Paul A. Kelly, David Gould, Hiroaki Okamura, Jean Dijane
Two techniques were routinely employed to detect the proteins separated by SDS-poly-aerylamide gel electrophoresis (PAGE) that is, silver staining and autoradiography of a gel of a radioiodinated-purified receptor. One faint band at Mr, ~32,000 was occasionally detected by Coomassie Brilliant Blue staining. However, silver staining detected at least nine major bands. Iodination of the affinity-purified receptor and analysis by SDS-PAGE also revealed several bands. When the iodinated receptor was repurified on a PRL-Affi-Gel column, only the Mr ~42,000 band was seen.50
Mechanisms of Fibril Formation and Cellular Response
Published in Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin, XIth International Symposium on Amyloidosis, 2007
Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin
37 °C for 1 h. CD spectra were obtained by using a JASCO J-720 spectropolarimeter at 25 °C. A molecular mass of the TTR of 14 kDa was used for calculation of the mean residue ellipticity. The fluorescence intensity of Trp was measured via a Hitachi F-4500 spectrofluorimeter at 25 °C. All assays used excitation at 295 nm and emission at 340 nm. Excitation and emission slits were set at 5 nm. To assess the amount of amyloid fibrils in vitro, thioflavin T test has been widely used. Fluorescence spectra were obtained by using a HITACHI F-4500 spectrofluorimeter with an assay volume of 1 ml. All assays used excitation at 450 nm and emission at 482 nm. Excitation and emission slits were set at 5 nm. The reaction mixture contained 5 uM thioflavin T and 50 mM Gly-NaOH buffer, pH 10.0. Nonboiled SDS-PAGE was performed under nondenaturing conditions. One microgram of the TTR samples incubated for 37 °C for 5 days at pH 3.0 as described above was neutralized with PBS to obtain a final pH greater than 6.5. After neutralization, samples were mixed with 5% SDS sample buffer and loaded on 15% polyacrylamide gels, which were stained with Coomassie Brilliant Blue. Intensities of the bands were evaluated by densitometric analysis using ATTO densito.
Cholesterol-linoleic acid liposomes induced extracellular vesicles secretion from immortalized adipose-derived mesenchymal stem cells for in vitro cell migration
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2023
Jzit Weii Chen, Fong Fong Liew, Hsiao Wei Tan, Misni Misran, Ivy Chung
Acetic acid, chloroform and hydrochloric acid were obtained from R&M Chemicals, Kuala Lumpur, Malaysia. Low-molecular-weight chitosan and cholesterol were obtained from Sigma-Aldrich (St. Louis, MO). Linoleic acid and DOTAP were obtained from Fluka (Buchs, Switzerland) and Avanti Polar Lipids (Alabaster, AL), respectively. Analytical grade sodium nitrate was obtained from HmbG Chemicals (Kuala Lumpur, Malaysia). Sodium hydroxide (reagent grade, ≥98%, pellets (anhydrous)) was obtained from Merck (Solna, Sweden). Low glucose Dulbecco’s modified Eagle medium (DMEM), foetal bovine serum (FBS), penicillin–streptomycin (Pen–strep) (10,000 U/ml) and GlutaMax were obtained from Gibco (Carlsbad, CA). Immortalized AD-MSCs, ASC52telo were obtained from America Type Culture Collection (ATCC) (Manassas, VA). Human keratinocytes (HaCaT) cell line and optimized DMEM were obtained from AddexBio (San Diego, CA). Pierce™ bovine serum albumin (BSA) standard (2 mg/ml) was obtained from Thermo Fisher Scientific (Waltham, MA). Cell culture falcon tubes, dishes and plates were obtained from NEST Biotechnology Co. (Wuxi, China). Dulbecco’s phosphate-buffered saline (DPBS) was obtained from Sangon Biotechnology (Shanghai, China). Minisart® syringe filter, surfactant-free cellulose acetate (SFCA) and pore size of 0.8 µm were obtained from Sartorius (Goettingen, Germany). Amicon® Ultra-15 centrifugal filter devices-10 kDa and exoEasy Maxi Kit were obtained from Merck (Darmstadt, Germany) and Qiagen (Hilden, Germany). Coomassie Brilliant Blue (CBB) dye was obtained from Nacalai Tesque Inc. (Kyoto, Japan).
Cytotoxic, necrotic, apoptotic, and autophagic properties of venom sac extract of Vespa orientalis in T47D and MCF10A breast cell lines
Published in Toxin Reviews, 2023
Seyed Mohammad-Hossein Shetab-Boushehri, Asieh Hosseini, Javad Rafinejad, Alireza Ebadollahi-Natanzi, Seyed Vahid Shetab-Boushehri
The protein content of the VSE of hornet was measured according to modified Bradford protein assay (Zor and Selinger 1996). Briefly, a 50 ml of 0.1 mg.ml−1 solution of coomassie brilliant blue G-250 in a mixture of 95% ethanol (2.5 ml), 85% orthophosphoric acid (5 ml), and distilled water (42.5 ml) was made and used as a reagent. A 200 µg.ml−1 stock solution of BSA in DW was made and serially diluted to make 10, 20, 30, 50, 70, 90, 100, 120, 140, 160, 180, and 200 µg.ml−1 working solutions of BSA in DW. To 5 µl of each standard protein solution or 100-fold diluted sample of VSE, 250 µl of coomassie brilliant blue reagent was added in each well of a 96-well plate. After 5 min, absorbances of solutions were measured at 450 nm and 590 nm by a multi-mode microplate reader (Synergy™ HT Microplate Reader, BioTek Instruments Inc., Winooski, Vermont, USA). The absorbance of DW as blank was subtracted from those of standards and the VSE sample. Subtracted absorbances of standards and VSE sample at 590 nm were then divided by respective absorbances at 450 nm. The protein standard (calibration) curve was constructed by depicting the 590 nm/450 nm absorption ratio of standards versus their different concentrations. Protein concentration of VSE was determined by interpolation of 590 nm/450 nm absorption ratio of VSE sample over tabulated standards concentrations on protein standard curve followed by multiplication by the dilution factor. The measurements were performed in triplicate samples.
MMP-3 activation is involved in copper oxide nanoparticle-induced epithelial-mesenchymal transition in human lung epithelial cells
Published in Nanotoxicology, 2021
Yuanbao Zhang, Yiqun Mo, Jiali Yuan, Yue Zhang, Luke Mo, Qunwei Zhang
MMP-3 activity was measured by β-casein zymography assay while MMP-2 and MMP-9 activity by gelatin zymography assay as described previously with modifications (Mo et al. 2019; Wan et al. 2011; Yamashita et al. 2011). Briefly, cells were seeded in 6-well plates in serum-free RPMI1640. After Nano-CuO or Nano-TiO2 treatment, the conditioned medium was collected. For β-casein zymography assay, the conditioned medium was concentrated ten times by Amicon® Ultra Centrifugal Filter with Ultracel-10K membrane (Millipore, Billerica, MA). Electrophoresis was performed on 10% SDS-polyacrylamide gel with 1 mg/mL β-casein (for MMP-3) or 0.5 mg/mL gelatin (for MMP-2 and MMP-9) under non-reducing conditions. After electrophoresis, the gels were washed twice (30 min each) in 2.5% Triton X-100 solution, and incubated in calcium assay buffer (pH 7.5) containing NaCl (150 mM), CaCl2 (10 mM), ZnCl2 (5 μM), and 1% Triton X-100 at 37 °C for 36 h. After staining with 0.1% Coomassie Brilliant Blue R-250 (Bio-Rad, Hercules, CA), the gels were destained in 10% acetic acid until clear bands were observed against the background of Coomassie blue-stained gel.