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Basic genetics and patterns of inheritance
Published in Hung N. Winn, Frank A. Chervenak, Roberto Romero, Clinical Maternal-Fetal Medicine Online, 2021
Performing cytogenetic analysis has become an integral part of many areas of medical practice, including obstetrics, pediatrics, and oncology. Traditional chromosome analysis first involves obtaining a sample of living tissue. This can be blood, skin, amniotic fluid, products of conception, bone marrow, or any viable solid tissue. Blood is the most frequently analyzed tissue for routine chromosome analysis. For blood, the lymphocytes are isolated. For amniotic fluid, the amniocytes are obtained by spinning down the fluid and removing the cell pellet. For solid tissues, the tissue is minced and/or sonicated. For all tissue types, the cells are cultured in tissue culture for 48 to 72 hours. Cell division is arrested at metaphase by the addition of colcemid. The cells are then harvested and placed on a microscope slide. The cell nuclei are ruptured by adding a hypotonic solution, then stained to show the bands, and images of the metaphase chromosome spreads are analyzed. Each chromosome is studied by looking at the banding pattern to identify not only numerical abnormalities, but also structural problems. Newer computerized technology allows karyotype analysis by digital imaging methods. By convention, the 22 autosome pairs are arranged by size, from the largest to the smallest, in four rows, with the pair of sex chromosomes in the lower right corner (Fig. 1). Ideograms are schematic representations of banding patterns used by cytogeneticists to standardize numbering of specific bands (Fig. 2).
Methods in Experimental Pathology of the Pleura
Published in Joan Gil, Models of Lung Disease, 2020
Routine cytogenetic methods are followed. Colcemid (0.01 μg) is added to the cells 48-72 hr after plating for 1-2 hr. The cells can be grown in a plastic flask or on a cover glass. The cells are processed in situ or detached with 0.25% trypsin, swollen with 75 mM KCl, and fixed with 3:1 methanolacetic acid three times. The spread out chromosomes are usually G-banded (Jaurand et al., 1986).
Tumor Cell Invasion In Vitro in Syngeneic and Heterologous Systems
Published in Rolf Bjerkvig, Spheroid Culture in Cancer Research, 2017
The chorioallantoic membrane (CAM) has been used as a target for tumor cells both in qualitative and quantitative studies of tumor cell invasion. Ambrose and Easty studied the cellular interactions of normal cells26 and malignant cells27 with CAM by use of time-lapse photography. Easty and Easty described an assay using chick chorioallantoic membrane from fertile eggs.28 The membrane was carefully removed from the egg and placed on a metal grid. A suspension of single cells was then seeded on the top of the membrane and incubated for 7 d. The authors were able to discriminate between invasive and noninvasive cells and quantify the invasion by measuring the distance of tumor cell penetration into the membrane. This assay was used to study the anti-invasive effects of dibutyryl cAMP and colcemid. Hart and Fidler29 used CAM to quantify invasion by using 125I-IdUrd-labeled cells that were added to an invasion chamber. Cells traversing the CAM were collected and quantified in a sponge situated under the membrane. A modification of this assay system has also been described, where the invading cells were collected in semisolid agar, allowing the cells to be used for further experiments.30 The CAM contains a variable number of host cells that may modify the invasive pattern of malignant cells. To more closely mimic the human basement membrane, human amnion denuded of epithelium by an alkaline solution31,32 has been used extensively to study the biological properties linked to tumor33–37 and normal cell invasion.38
Evaluation of toxicological and antimicrobial activity of lavender and immortelle essential oils
Published in Drug and Chemical Toxicology, 2021
Aner Mesic, Irma Mahmutović-Dizdarević, Emina Tahirović, Irma Durmišević, Izet Eminovic, Anesa Jerković-Mujkić, Renata Bešta-Gajević
The chromosome aberrations (CAs) test was modified according to Moorhead et al. (1960). Lymphocyte cell cultures were incubated at 37 °C for 72 h. The test concentrations of lavender and immortelle EOs were added to lymphocyte cultures after 48 h of incubation at the following final concentrations: 0.10; 0.20, and 0.30 µl/ml and the cultures incubated for a further 24 h. The test concentrations of lavender and immortelle oils were prepared with 0.1% DMSO and RPMI-1640 medium. In order to arrest the cells at the metaphase stage, at 70 h of incubation, colcemid™ solution (0.2 µg/ml) (Sigma-Aldrich, St. Louis, MO) was added. In all of the experiments, DMSO (0.1%) was used as the solvent control, while untreated culture was the control group. After an incubation period of 72 h, the cells from culture were treated with a hypotonic solution (0.075 M KCl) for 30 min at 37 °C, to lyse the red blood cells, and fixed with a cold methanol/glacial acetic acid fixative (3:1 v/v). The fixation included three changes of the fixative. Pre-fixed lymphocytes were spread onto clean and cooled glass slides, air-dried, stained with 10% Giemsa solution and analyzed under 1000x magnification.
Toxic and irritant effects induced by zearalenone: prevention by taurine
Published in Toxin Reviews, 2021
500 µL of the blood sample, TU (50 μM), ZEN (10, 20 and 40 μM) and their combinations were added to culture medium containing 10 µg/mL of 5-bromo-20-deoxyuridine (BrdU; Sigma), 7 ml of Chromosome Medium B (Biochrom, Leonorenstr. 2–6.D-12247, Berlin), 100 U/mL penicillin, 100 µg/ml streptomycin and 0.5 ml of phytohemagglutinin (Biochrom) and cell culture was incubated at 37 °C for 72 h. At 70 h of incubation, 0.06 µg/mL of colcemid (Sigma) which is a mitotic inhibitor was added in the culture medium. At the end of the incubation period, the culture medium was centrifuged at 900 × g for 10 min and lymphocytes cell obtained were hypotonized by 0.075 M of cold potassium chloride for 30 min and then cells were fixed with ice‐cold methanol/acetic acid (3:1, v/v). Microscope slides were assayed as triplicate using the standard procedure, air-dried, and stained according to fluorescence plus Giemsa (FPG) as described by Perry and Wolff (1974). To scoring of SCE cell, 25 well-spread second division metaphases containing 46 (± 2) chromosomes were counted) using a light microscope at 1000 x magnification by a single observer. 1000 metaphases in total were counted for each concentration and the results were calculated as SCE per cell.
Determination of spontaneous dicentric frequencies and establishment of dose-response curves after expose of human peripheral blood lymphocytes to low- and high-dose rate 60Co gamma rays – the basis for cytogenetic biodosimetry in Vietnam
Published in International Journal of Radiation Biology, 2019
Ngoc-Duy Pham, Minh-Hiep Nguyen, Que Tran, Quang-Tuan Che, Van-Hung Nguyen, Van-Toan Phan, Van-Dung Pham, Suen Ern Lee, Thi-Linh-Tien Vo
Lymphocyte cell culture was carried out according to ISO 19238 and IAEA standard protocols (ISO 19238 2014; IAEA 2011). Briefly, 0.6 ml of whole blood was added into 5.4 ml completed RPMI-1640 medium (Sigma Aldrich) supplemented with 15% heat inactivated FBS (Invitrogen), 1.5% PHA (Invitrogen) and Kanamicine sulfate (Invitrogen), and then incubated in humidified air at 37 °C, 5% CO2 for 48 h. To control the first mitosis metaphases, the two cultures were conducted in parallel. In culture-I, colcemid (0.05 µg/mL) was added at the initiation of the culture. In culture-II, colcemid (0.3 µg/mL) was added 2 h before harvesting. The number of diploid cells (2n) and polyploid cells (4n) in culture-I were counted. When the proportion of polyploid cells was higher than 5%, the slides prepared in culture-I were used and only haploid (2n) metaphases were scored for chromosome aberrations. When the proportion of polyploid cells was lower than 5%, we scored the slides prepared in culture-II.