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Bone Marrow Cell Counting: Methodological Issues
Published in Adrian P. Gee, BONE MARROW PROCESSING and PURGING, 2020
Elizabeth J. Read, Charles S. Carter, Herbert M. Cullis
Although other assays, such as hematopoietic colony assays and surface marker phenotyping by flow cytometry, are available for further characterize the cellular content and offer more detailed information on the hematopoietic progenitor content of the marrow suspension, cell counting remains an essential part of the quality control program for bone marrow processing. Of all assays, cell counting provides the most rapid feedback to laboratory and clinical personnel. In some institutions, a midharvest cell count is obtained to provide an estimate of how much more harvesting is required. In syngeneic and allogeneic marrow transplantation, as well as for some autologous protocols, infusion of the marrow is almost always done with cell counts being the only laboratory assay available, because accurate quantitation of hematopoietic progenitor colonies requires 7 to 14 days.
Reproductive Biotechnologies Applied to Artificial Insemination in Swine
Published in Juan Carlos Gardón, Katy Satué, Biotechnologies Applied to Animal Reproduction, 2020
Francisco Alberto García-Vázquez, Chiara Luongo, Gabriela Garrappa, Ernesto Rodríguez Tobón
Once the ejaculate is suitable for processing after this initial appreciation, the next step is the determination of spermatozoa concentration. Traditionally, the most used method to calculate the cell concentration has been the counting chambers such as Neubauer, Bürker, and Thoma (Althouse, 2007; Brito et al., 2016). Nowadays, this method has been surpassed by spectrophotometric techniques because manual counting is a slow process (Brito et al., 2016). In an ejaculate, the opacity depends on the number of cells among other elements of the SP that could interfere with the passage of the light through the sample. Therefore, it is recommendable to dilute a small sample for obtaining a more reliable result (depending extender, dilution rates from 1:4–1:25) (Althouse, 2007). Besides, errors in the evaluation can occur if the dilution is not correct or untimely reading (Althouse, 2007; Brito et al., 2016). CASA (Computerized Assisted Sperm Analysis) system can also be used to determine the concentration, although this system is more recommendable to assess sperm motility (Amann and Waberski, 2014; Brito et al., 2016), as will be explained later. Another method for cell counting is the flow cytometry, which allows rapid and automated counts of many cells. However, the use of flow cytometry is limited due to its high cost and the need for qualified personnel to manage the equipment and interpret the results (Brito et al., 2016).
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.
Heat-treated human platelet pellet lysate modulates microglia activation, favors wound healing and promotes neuronal differentiation in vitro
Published in Platelets, 2021
Ouada Nebie, Lassina Barro, Yu-Wen Wu, Folke Knutson, Luc Buée, David Devos, Chih-Wei Peng, David Blum, Thierry Burnouf
SH-SY5Y, EA-h926, and BV-2 cells were seeded in 96-well plates at a density of 104 cells/mL and incubated for 24 h to allow attachment. They were then treated with 5% (v/v) HPPL or 5% I-HPPL and incubated for an additional 24 h. Control cells were treated with DMEM supplemented with 5% or 10% FBS. Cell viability was quantified using a cell counting kit (CCK-8, Sigma, St. Louis, MO, USA). Ten microliters of the CCK-8 reagent solution were added to each well and incubated for 4 h, and the absorbance was measured at 450 nm using a 96-well microplate reader on a spectrophotometer (Infinity M200, Tecan, Männerdorf, Switzerland). The absorbance of untreated controls was considered 100% survival, and that of treated cells was taken as a percentage of viable cells relative to the control. The background optical density (OD) value was taken from wells without cells but containing supplements also incubated with the CCK-8 solution. The effect of treatment on cell viability was presented for each concentration as the mean ± standard deviation (SD) determined from at least three independent experiments.
Instrument-based Tests for Measuring Anterior Chamber Cells in Uveitis: A Systematic Review
Published in Ocular Immunology and Inflammation, 2020
Xiaoxuan Liu, Ameenat L. Solebo, Livia Faes, Sophie Beese, Tasanee Braithwaite, Matthew E. Round, Jesse Panthagani, Aditya U. Kale, Thomas W. McNally, Didar Abdulla, Pearse A. Keane, David J. Moore, Alastair K. Denniston
Two reviewers extracted data independently using a pre-specified data extraction sheet. The data included population characteristics (number of participants, gender, age, underlying etiology), index test characteristics (technology, manufacturer, model, image acquisition settings, area sampled and software automation), clinician grading (name of grading system used, number of patients in each grade) and outcome (correlation coefficient, inter/intra-observer reliability). Cell counting analysis was recorded as fully automated, semi-automated or manual. For the clinician grading, we extracted how each grade was defined and whether any modifications were made to validated clinical grading systems. We contacted three authors for further information16–18, all of whom responded and one provided further data (confidence intervals) which was not reported in the original paper.16
Characterization and cytotoxicity assessment of nargile smoke using dynamic exposure
Published in Inhalation Toxicology, 2019
Christian Khalil, Joe Braham Chahine, Brenda Chahla, Tamara Hobeika, Rony S. Khnayzer
Cell membrane damages were assessed immediately post exposure using the Countess® Automated Cell Counter (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The Tryptan blue assay was based on the dye exclusion principle whereby cells with intact cell membranes will exclude the Tryptan blue dye while dead cells do not. The cells were lifted from the Snapwell inserts using a Trypsin solution. The trypsin was neutralized-using media before mixing with Tryptan blue and adding to the cell counting slides from Invitrogen for assessment. Briefly, 10 μL of Tryptan blue was mixed with 10 μL cellular suspension and 10 μL of the resulting mixture was pipetted into the slide before inserting into the countess apparatus for cell viability determination and counting. This methodology generated a number of parameters including total cell count, percentage viable cells, and percentage dead cells. The use of automated evaluation was determined as efficient alternative for cell counting when handling large number of samples as per previous investigations (Cadena-Herrera et al. 2015).