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Assessment of Airway Smooth Muscle Growth and Division: In Vitro Studies
Published in Alastair G. Stewart, AIRWAY WALL REMODELLING in ASTHMA, 2020
Although this is a very simple technique, it is very sensitive to operator error. The subjective nature of cell counting can make it difficult to obtain reproducible counts within the haemacytometer chamber unless a brief “running-in” or learning period is allowed by the operator. Care should also be taken to count only living cells. A minimum of 120–140 viable cells should be counted to limit the statistical error associated with counting. The major disadvantage of this technique, however, is that it is labourious and time-consuming, and only relatively small numbers of samples can easily be processed. Some of these limitations are overcome by electronic cell counting (e.g., with a Coulter counter, Miami, FL, USA), but it is still considerably slower than automated colourimetry and may underestimate some cytotoxic effects because it estimates cell number on the basis of size rather than metabolic activity.
Synovial Fluid
Published in Verna Wright, Eric L. Radin, Mechanics of Human Joints, 2020
Pierre Geborek, Frank A. Wollheim
A fair estimation of cellular content can be achieved simply by applying a drop of SF to a slide and directly examining it under a microscope. It is usually easy to classify the SF as either inflammatory or noninflammatory. For more precise information on cell concentration, a hemocytometer chamber is used. Differential count is best done on dried and stained smears, although with some experience a poly-mono ratio can be done by using a phase-contrast device directly in the hemocytometer chamber. Identification of cells other than mononuclear cells and neutrophils yields little diagnostic information, although eosinophils, mast cells, ragocytes, Lupus erythematosus (LE)-cells, platelets, and others have been described (3,6). Occasionally malignant cells can be identified in SF.
Assay of Antibiotics in Mammalian Cell Culture
Published in Adorjan Aszalos, Modern Analysis of Antibiotics, 2020
The counting of cell populations with the aid of a hemocytometer and microscope has had wide application in tissue culture; for small numbers or volumes of samples it is the method most commonly used. This method has the distinct advantage over electronic enumeration, as quantitative information on cell viability may be obtained simultaneously by the incorporation of a vital stain into the diluting medium. However, this method quickly becomes tedious in the counting of large numbers of samples, a factor likely to increase the existing counting error, estimated at 10% [10]. Automated electronic counters, which have been widely used in recent years for studies on growth and population dynamics, offer greater rapidity and accuracy and their application to mammalian cells is well defined [11]. With sufficienty large populations of cells that can be readily dissociated in the absence of cell damage or debris formation, it is clearly the method of choice for quantitative growth studies.
Galangin ameliorates experimental autoimmune encephalomyelitis in mice via modulation of cellular immunity
Published in Journal of Immunotoxicology, 2021
Kok-Tong Tan, Shiming Li, Lauren Panny, Chi-Chien Lin, Shih-Chao Lin
At necropsy, the spleen of each mouse was aseptically-harvested and single-cell suspension prepared. In brief, each spleen was filtered through a sterile 50-mesh stainless steel mesh and then red blood cells present were lysed using commercial RBC lysing buffer (Sigma). After washing by centrifugation, the splenocytes were re-suspended in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 50 μg gentamicin/ml (all ThermoFisher Scientific, Waltham, MA), as well as with 2-mM glutamine (Sigma), and 50-μM 2-mercaptoethanol (Amresco, Cleve- land, OH, USA). Cell concentrations were determined in a hemocytometer. Thereafter, aliquots of splenocytes were placed in 6-well plates (2 × 106/well) and received either medium or were stimulated by addition of 25 µg MOG 35–55/ml for 3 d at 37 °C. At the end of this period, culture supernatants were collected and levels of cytokines (TNFα, IL-6, IL-12 p70) and chemokines (MCP-1, MIP-1α) in the medium were analyzed using specific anti-mouse ELISA kits from PeproTech (London, UK).
An overview of experiments with lead-containing nanoparticles performed by the Ekaterinburg nanotoxicological research team
Published in Nanotoxicology, 2020
Ilzira A. Minigaliyeva, Marina P. Sutunkova, Vladimir B. Gurvich, Tatiana V. Bushueva, Svetlana V. Klinova, Svetlana N. Solovyeva, Ivan N. Chernyshov, Irene E. Valamina, Vladimir Y. Shur, Ekaterina V. Shishkina, Oleg H. Makeyev, Vladimir G. Panov, Larisa I. Privalova, Boris A. Katsnelson
A single-shot intratracheal instillation of 0.2 mg PbO-NP in 1 mL water suspension (or of sterile de-ionized water from the same batch without any particles) served as an experimental model for the response of the lower airways to particle deposition. To assess this response 24 h later, a cannula connected to a Luer syringe containing 10 mL of normal saline was inserted into the surgically prepared trachea of a rat under hexenal anesthesia. The fluid entered the lungs slowly under the gravity of the piston, with the animal and syringe positioned vertically. Then the rat and the syringe were turned 180°, and the fluid flowed back into the syringe. The extracted broncho-alveolar lavage fluid (BALF) was poured into siliconized refrigerated tubes. An aliquot sample of the BALF was drawn into a WBC count pipette together with 3% acetic acid and methylene blue. Cell count was performed in a standard hemocytometer (the so-called Goryayev’s Chamber). For cytological examination, the BALF was centrifuged for 4 min at 1000 rpm, then the fluid was decanted, and the sediment was used for preparing smears on two microscope slides. After air drying, the smears were fixed with methyl alcohol and stained with azure eosin. The smears were observed under a microscope with immersion at a magnification of 1000×. The differential count for determining the percentage of alveolar macrophages (АМ), neutrophil leucocytes (NL), and other cells counted together were conducted up to a total number of 100 cells. Allowing for the number of cells in the BALF, these percentages were recalculated in terms of absolute AM and NL counts.
3,3′-Diindolylmethane Inhibits TNF-α- and TGF-β-Induced Epithelial–Mesenchymal Transition in Breast Cancer Cells
Published in Nutrition and Cancer, 2019
MCF-7 and HCC38 (3 × 103 cells/well) in maintenance medium were seeded into respective 96-well plates. After 24 h of maintenance, the cells were treated with DIM and/or TNF-α/TGF-β in serum-free medium for 24 h. The cell proliferation assay was performed with the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The viability of the cells was determined by trypan blue dye exclusion assay. In brief, the MCF-7 cells (7 × 104 cells/well) in maintenance medium were seeded into respective 12-well plates. After 24 h of maintenance, the cells were treated with DIM and/or TNF-α/TGF-β in serum-free medium for 24 h. Viable cells were counted using a hemocytometer.