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Systemic Lupus Erythematosus
Published in Jason Liebowitz, Philip Seo, David Hellmann, Michael Zeide, Clinical Innovation in Rheumatology, 2023
Vaneet K. Sandhu, Neha V. Chiruvolu, Daniel J. Wallace
Additional technological advances include an array-based multiplex ELISA system that has been used to analyze urine for 1,000 proteins. Phase 1 trials were able to identify multiple proteins in the urine of patients with LN that were not found in the urine of healthy controls.14 Separately, CYTOF mass cytometry (CyTOF) is used for comprehensive multidimensional single-cell phenotyping that uses rare earth metal isotope tags instead of fluorescent-tagged antibodies. One study using this technology showed significant differences between circulating immune cells in blood compared to healthy controls. They found high numbers of activated CD57+ CD8 T cells and reduced NK cell percentages in SLE patients.14
Time to ‘Couple’ Redox Biology with Exercise Immunology
Published in James N. Cobley, Gareth W. Davison, Oxidative Eustress in Exercise Physiology, 2022
Alex J. Wadley, Steven J. Coles
The development of CyTOF™ mass cytometry may be a solution to current experimental limitations. This technology has permitted high-resolution assessment of natural killer/T cell immunophenotype and functional analysis at the single-cell level (Kay, Strauss-Albee and Blish, 2016; Brodie and Tosevski, 2018). Like traditional flow cytometry, CyTOF™ mass cytometry utilises specific antibodies (labelled with metals in this instance) to detect the expression of various cellular antigens (Heck, Bishop and Ellis, 2019). Because the technology is based on time-of-flight mass spectrometry as a detection method, the limitations/complications associated with standard flow cytometry (e.g., spectral overlap) are ameliorated. Therefore, upwards of 50 different immunophenotypic and(or) functional molecules for a single cell may be detected simultaneously. So far protocols have been established to concurrently evaluate cytokines, transcription factors and immunophenotypic markers (Lin, Gupta and Maecker, 2015; Simoni et al., 2018). In theory, the analysis of protein/peptide markers of the intracellular redox environment (e.g., peroxiredoxin, thioredoxin, glutaredoxin, glutathione) as well as redox-sensitive transcription factors implicated with oxidative and reductive stress (Bellezza et al., 2018) could easily be incorporated into CyTOF™ mass cytometry workflows.
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).
Sulfoxaflor insecticide exhibits cytotoxic or genotoxic and apoptotic potential via oxidative stress-associated DNA damage in human blood lymphocytes cell cultures
Published in Drug and Chemical Toxicology, 2023
Cebrail Sınacı, Ayla Çelik, Derya Yetkin, Sertan Çevik, Gizem Güler
In the flow cytometry, cells or particles in the liquid are passed through a chamber illuminated by laser light, and the stimuli given by the cells as they pass through the light are collected and analyzed. Here, the cells' physical properties such as size and granularity can be shown; antigenic structures on the cell surface or content can also be determined. Thus, information can be obtained about various properties of the cell, such as immune structure, DNA content, enzyme activity, cell membrane potential, and viability and apoptotic features of cells (Dunphy 2004, Atalay et al. 2018). It has been shown that it can analyze much more cells in a shorter time compared to light microscopy, and with this technique, an average of 10 000 cells can be evaluated in 20 seconds (Pozarowski et al. 2004, Toduka et al. 2012).
Single-cell RNA sequencing: An overview for the ophthalmologist
Published in Seminars in Ophthalmology, 2021
Elizabeth J. Rossin, Lucia Sobrin, Leo A. Kim
One of the most common approaches for classifying cell type is fluorescence-activated cell sorting (FACS), which can be used to identify and sort sub-populations of cells based on size, morphology and surface proteins with the use of fluorescently conjugated antibodies. These sub-populations can be further characterized by pooled RNA sequencing or functional assays. However, marker-based approaches are inherently constrained by the availability and choice of markers and by our knowledge of how markers define cell types. Even within a seemingly narrowly defined group of cells based on cell surface markers, there is likely heterogeneity in gene expression signature.3 More recently, mass cytometry has been employed, which involves cell characterization with antibodies labelled by heavy metal ions, and this has dramatically increased the number of proteins that can be assessed at one time by five to 10-fold.4 Still, it is challenging to assess the entire proteome all at once with flow cytometry.
Priming of pancreatic cancer cells with bispecific antibody armed activated T cells sensitizes tumors for enhanced chemoresponsiveness
Published in OncoImmunology, 2021
Archana Thakur, Johnson Ung, Elyse N. Tomaszewski, Amy Schienschang, Timothy M. LaBrie, Dana L. Schalk, Lawrence G. Lum
Flow Cytometry and Imaging Flow Cytometry. Single cell suspensions of MiaPaCa-2 cell line and its CIS- and GEM-resistant derivatives grown in 2D culture or 3D tumorspheres were stained with the mouse anti-human fluorescence-labeled antibodies: EpCAM-APC-Cy7, CD44-FITC, CD24-APC, and HER2-PE (BD Biosciences) to analyze the proportion of % positive CSC by flow cytometry. For imaging flow cytometry, EpCAM+/EGFR+ cells were sorted to stain with CD44-FITC, CD24-PerCp Cy5.5 (Red) and HER2-PE (yellow) to analyze on ImageStreamX System. Parental cell lines and their CIS- and GEM-resistant derivatives were also stained for the expression of efflux drug transporters with the following anti-human fluorescence-labeled antibodies: ABCB1 (MDR-1)-AF647, ABCC1 (MRP-1)-AF488, ABCC5-AF405 and ABCG2-PE. Cells were analyzed on a FACScalibur (BD Biosciences) or NovoCyte, and the data was analyzed using FlowJo software (BD Biosciences) or NovoExpress (Agilent Technologies). The acquired data was analyzed using the IDEAS (v6.1, Amnis) software.