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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.
Image Analysis for Tissue Phenomics
Published in Gerd Binnig, Ralf Huss, Günter Schmidt, Tissue Phenomics, 2018
Johannes Zimmermann, Keith E. Steele, Brian Laffin, René Korn, Jan Lesniak, Tobias Wiestler, Martin Baatz
Using image analysis, tissue phenomics is well suited to address complex biological questions. For instance, it can utilize highly multiplexed tissue techniques such as immunofluorescence, co-registered serial IHC, or imaging mass cytometry together with image analysis in a variety of ways. These kinds of multifaceted approaches can add great value to the data needed to optimize tissue phenomics. Complexity, however, also increases the potential to introduce noise into a system. Sources of noise must, therefore, be minimized. In short, image analysis is best when the experimental design and its execution maximize the extraction of all potentially quality data and at the same time minimize the effect of potential sources of problematic data. However, it is not necessarily obvious and straightforward what the experimental design and execution plan should be. In that regard, some pre-testing, validation, and, if needed, a revision may be required before settling on the final elements of the experimental design. We briefly discuss a few points to illustrate how design elements can influence the success of the image analysis study, as follows.
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.
Intestinal microbial communities and Holdemanella isolated from HIV+/− men who have sex with men increase frequencies of lamina propria CCR5+ CD4+ T cells
Published in Gut Microbes, 2021
Eiko Yamada, Casey G. Martin, Nancy Moreno-Huizar, Jennifer Fouquier, C. Preston Neff, Sara L. Coleman, Jennifer M. Schneider, Jonathan Huber, Nichole M. Nusbacher, Martin McCarter, Thomas B. Campbell, Catherine A. Lozupone, Brent E. Palmer
All participants were asked to prepare their bowel for biopsy using a Fleet Saline enema. Following the enema, 30 pinch biopsies were collected from the rectal region of the colon, approximately 3–10 cm from the anal verge. Two of these pinches were preserved for 16S rRNA targeted sequencing as described below and 23 of these pinches were used for CyTOF. The pinches were digested for 1.5 hours with DNase and collagenase and then filtered through a 70 μm nylon filter as previously described.2 Biopsy samples were immediately stained for mass cytometry. For live-dead cell distinction, cells were stained with 2.5 μM cisplatin (Fluidigm, South San Francisco, CA). Cells were then resuspended in 70 μl of barium-free FACS buffer (PBS with 0.1% BSA and 2 mM EDTA) and incubated for 30 min at 4°C with a 30 μl mixture of metal-conjugated antibodies (Abs) focused on T cells. Cells were stained with a DNA intercalator (0.125 μM 191Ir and 193Ir; Fluidigm) and acquired using a CyTOF2 mass cytometer (Fluidigm), CyTOF software v.6.0.626 with noise reduction, a lower convolution threshold of 200, event length limits of 10–150 pushes, a σ value of 3, and a flow rate of 0.045 ml/min. These data were analyzed using FlowJo and the frequencies of CD4+ CCR5+ were used to identify taxa that were positively correlated.
Approaching complexity: systems biology and ms-based techniques to address immune signaling
Published in Expert Review of Proteomics, 2020
Joseph Gillen, Caleb Bridgwater, Aleksandra Nita-Lazar
One of the earliest methods that uses mass spectrometry to analyze single cells is mass cytometry, also known as cytometry by time-of-flight or CyTOF. There are many excellent reviews of CyTOF that explain the mechanisms, benefits, and concerns involved in these experiments (reviewed in [54–56]). Briefly, specific targets are labeled with heavy metal tags then the single cells isolated and atomized using argon plasma. Then using the sensitivity and accuracy of an elemental time-of-flight mass spectrometer to detect the heavy metal tags, CyTOF can quantify up to 40 possible elements at a single cell level. While the measurement of 40 individual elements is an improvement over the number of elements that can be simultaneously measured with fluorophores (a process limited to less than 20 simultaneous measurements), the method of does have drawbacks. First, CyTOF uses atomization of the cells with argon plasma to free the metal markers for detection and cannot be used to collect live cells and the sensitivity for detection of the mass reporters prevents the detection of extremely rare molecules.