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Optimal Detection of and Effect of Vitamin D3 on Extrachromosomal Oncogene Sequences
Published in Maryce M. Jacobs, Vitamins and Minerals in the Prevention and Treatment of Cancer, 2018
Daniel D. Von Hoff, Donald R. VanDevanter
2. Alkaline Lysis. HL60 cells (2.0 × 107 cells per tube) and C5R500 cells (4.0 × 106 cells per tube) were lysed under alkaline conditions to isolate circular DNA.11 DNA pellets were rinsed once with ice-cold 70% ethanol, dried briefly, and suspended in 20 μl of 50mM sodium acetate, lOOmM NaCl, 1mM ZnSO4, pH 4.6 (S1 nuclease buffer).
Genetic analysis of the embryo
Published in David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham, Textbook of Assisted Reproductive Techniques, 2017
Yuval Yaron, Liran Hiersch, Veronica Gold, Sagit Peleg-Schalka, Mira Malcov
Lysis of the single embryonic cells and exposure of their genetic material to the PCR reagents is one of the most criti- cal steps, and greatly affects ADO rates and the efficiency and reliability of PGD (18). Among several options, the three most commonly used lysis solutions are water, alkaline lysis buffer, and proteinase K/sodium dodecyl sulfate (SDS) buf- fer. There is as yet no consensus as to which is superior. Water
Distribution and Toxicity of Retroviral Vectors after Intracavitary Delivery in Mouse and Man
Published in Eric Wickstrom, Clinical Trials of Genetic Therapy with Antisense DNA and DNA Vectors, 2020
Patrice S. Obermiller, Carlos L. Arteaga, Jeffrey T. Holt, Anne M. Pilaro
The retroviral vector was prepared by transfecting PA317 cells with the XM6:anti-fos retroviral vector DNA, which had been purified by alkaline lysis and ultracentrifugation twice on CsCl gradients. Following transfection, the PA317 cells were split and then treated with G418 until individual clones could be identified and expanded. Each clone was then assayed for vector titer by analyzing the supernatant's ability to transfer G418 resistance to MCF-7 cells. The clones which had the highest titer of vector production were then frozen in numerous aliquots and tested for sterility, presence of replication-competent retroviral vector, and presence of mycoplasma (Cornetta et al., 1993). The media from the XM6:anti-fos retroviral producer clone passed the following quality control tests: MAP test (mouse antibody production), in vitro test for adventitious viruses, bacterial and fungal sterility testing, mycoplasma testing, southern blot analysis of the retroviral producer clone, ability to transfer vector DNA to other cells (analyzed by transfer of G418 resistance and by southern blotting of target cells to demonstrate efficient gene transfer). Because distribution is only meaningful if replication competent vector is not present, we performed a PG-4 S+L- assay and 3T3 amplification followed by a PG-4 S+L- assay. Cells were tested for replicative retroviral contamination both by co-culture of producer cells and by amplification of retroviruses in supernatants by culture with Mus dunni cells. These tests, which included appropriate controls, showed no detection of replication competent viruses.
Dexamethasone as an anti-cancer or hepatotoxic
Published in Toxicology Mechanisms and Methods, 2023
Farzaneh Motafeghi, Parham Mortazavi, Nasrin Ghassemi-Barghi, Mohammad Zahedi, Mohammad Shokrzadeh
For the comet test, the slides were coated with 1% NMP (normal melting agarose) agarose, then the cells exposed to different concentrations of the substance were mixed with 1 mL of 1% LMP (low melting agarose), and a layer was placed on the coated slides and kept at 4 °C for 10 min. The slides were first immersed in an alkaline lysis buffer (pH = 10.0) to remove proteins and cell membranes. After that, they were placed in an electrophoretic alkaline buffer (pH > 13) for 40 min to allow the DNA to unwind. The same buffer was then electrophoresed for 40 min at 300 mA and 25 V with the same buffer. The slides were then removed and soaked in a neutralizing tris buffer solution (pH = 7.5) for 15 min before being stained with ethidium bromide (20 g/ml) for 10 min. Using a fluorescence microscope with a magnification of 400 and an excitation filter of 510–560 nm, and a barrier filter of 590 nm, examine the slides. The comet score software was used to calculate DNA damage, expressed as (tail length, % DNA in Tail, and Tail Moment), with the results given as mean (Shokrzadeh et al. 2020).
Comet Assay analysis of DNA strand breaks after exposure to the DNA-incorporated Auger Electron Emitter Iodine-125
Published in International Journal of Radiation Biology, 2023
Marcus Unverricht-Yeboah, Kathrin Holtmann, Ralf Kriehuber
Triton X-100 (1:100) and DMSO (1:10) were freshly added to the alkaline lysis solution (2.5 M NaCl, 100 mM Na3EDTA, 10 mM Tris Base, pH 10). For cell lysis, the slides with embedded cells were submerged in the alkaline lysis solution and incubated for 20–24 h at 4 °C. The slides were washed twice with 4 °C cold alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na3EDTA) and then incubated for 40 min with alkaline electrophoresis buffer at 4 °C. Then, electrophoresis was done at 1 V/cm for 30 min on ice in fresh alkaline electrophoresis buffer. Slides were then rinsed for 10 min in neutralization buffer (0.4 M Tris Base, pH 7.5) and incubated for 5 min in 70% ethanol. After drying for 10–15 min at 37 °C, the DNA was stained with propidium iodide (PI, 0.5 µg/ml in distilled water) for 30 min in the dark at RT. After rinsing the slides gently with water and drying by 37 °C, the slides with the embedded lysed cells were analyzed.
Sesamol Augments Paclitaxel-Induced Apoptosis in Human Cervical Cancer Cell Lines
Published in Nutrition and Cancer, 2022
Jiayi Xiong, Juanjuan Sheng, Yan Wei, Zhimin Sun, Xia Xiao, Lanmei Zhang
The oxidative DNA damage was analyzed by single-cell electrophoresis under the alkaline condition to understand the levels of single and double-strand breaks during paclitaxel alone and sesamol + paclitaxel treatment. Briefly, the treated HeLa cells were mixed with 100 μl of low melting agarose (0.5%), and the cell suspension was pipetted onto 1% standard melting agarose on microscope slides. Then, the microscopic slides were placed in cold lysis alkaline solution at pH 10 with 1% Triton X-100 and 10% DMSO at 4 °C for 1 h,. After alkaline lysis, the slides were placed in electrophoresis buffer (pH 13) for 20 min, and electrophoresis was performed (25 V) for 25 min. Then, the microscopic slides were neutralized using 0.4 M Tris at pH 7.5 for a short time (5 min). Finally, the slides were stained with 20 μg/ml of ethidium bromide to observe the presence of a comet tail. The images were analyzed to calculate % tail DNA using CASP software.