Cytometry Part A最新文献

Cytometry is a single cell, high-dimensional, high-throughput technique that is being applied across a range of disciplines. However, many elements alongside the data acquisition process might give rise to technical variation in the dataset, called batch effects. CytoNorm is a normalization algorithm for batch effect removal in cytometry data that was originally published in 2020 and has been applied on a variety of datasets since then. Here, we present CytoNorm 2.0, discussing new, illustrative use cases to increase the applicability of the algorithm and showcasing new visualizations that enable thorough quality control and understanding of the normalization process. We explain how CytoNorm can be used without the need for technical replicates or controls, show how the goal distribution can be tailored toward the experimental design and we elaborate on the choice of markers for CytoNorm's internal FlowSOM clustering step.

We have developed a 37-color spectral flow cytometry panel to assess the phenotypical differentiation of innate and adaptive immune lymphoid subsets within human intestinal tissue. In addition to lineage markers for identifying innate lymphoid cells (ILC), TCRγδ, MAIT (mucosal-associated invariant T), natural killer (NK), CD4⁺ and CD8⁺ T cells, we incorporated markers of differentiation and activation (CD45RA, CD45RO, CD25, CD27, CD38, CD39, CD69, CD103, CD127, CD161, HLA-DR, CTLA-4 [CD152]), alongside transcription factors (Bcl-6, FoxP3, GATA-3, Helios, T-bet, PU.1 and RORγt) and chemokine receptors (CCR4, CCR6, CCR7, CXCR3, and CXCR5). Additionally, Granzyme B and Ki-67 were included to assess cytotoxicity and proliferation potential of the different subsets. This panel is currently used for in-depth immunophenotyping in endoscopic biopsies and peripheral blood mononuclear cells (PBMC) from inflammatory bowel disease (IBD) patients. Distinguished from other OMIP papers, the comprehensive detection of both transcription factors and chemokine receptors facilitates the efficient assessment of several subsets, particularly CD4⁺ T helper cells, and its potential application extends to both tissue and circulation.

An emulsion of silicone oil droplets in aqueous buffer produces a distinctive series of peaks or resonances in the side scatter histogram in a flow cytometer. As many as 12 peaks are observed in the violet-side scatter channel at 405 nm, with half that number observed in the blue side scatter channel at 488 nm. Using the index of refraction of the oil and buffer, the wavelength of light, and the collection angle and gain of the instrument, we assign the peaks to specific diameters at which Mie resonances occur. With the close match for the index of refraction of silicone oil (n = 1.417 at 405 nm) to biological materials, these resonances could form the basis of a finely spaced size calibration ladder in the range 0.5-6 μm for estimating the size of biological particles in a flow cytometer. Resonances were also observed using mineral oil (n = 1.483 at 405 nm) suggesting that investigating and modeling resonances for emulsion systems may be useful for understanding these systems.

Identifying factors that contribute to the transition to the dilated phase in cardiac ischemia is a critical challenge in heart failure treatment. Currently, no effective therapies exist for this ischemic complication, and the mechanisms driving left ventricular dilatation during chronic post-infarction remodeling remain poorly understood. One potential pathological process leading to ventricular dilatation involves specific compensatory rearrangements in the border zone adjacent to the infarct, which isolates the intact myocardium from inflammation at the scar edge. Using a rat model, we examined ultrastructural changes in the intact and border zones of post-infarction myocardium at chronic stages. Morphometric analysis of myofibrils, mitochondria, and excitation-contraction coupling structures revealed similar remodeling processes in both zones at 2 weeks post-infarction, characterized by decreased myofibril density, reduced mitochondrial area and volume density, and shortened contacts between T-tubules and sarcoplasmic reticulum. At 26 weeks post-infarction, during the dilated cardiomyopathy phase, we observed distinct compensatory changes in the border zone. Specifically, there was a loose arrangement of myofibrils and an increased volume fraction of mitochondria. These differences in remodeling between the intact and border zones highlight factors contributing to ventricular dilatation and help the development of new therapeutic strategies to delay heart failure progression in cardiac ischemia.

Mycoplasma hyorhinis is a frequently observed contaminant in cell cultures, and its detection and purification pose considerable challenges. Fragments or other cell components are similar in size to those of Mycoplasma; therefore, distinguishing them is difficult. In this study, we used Hoechst staining in combination with carboxyfluorescein succinimidyl ester (CFSE) to label Mycoplasma. The trigger threshold was set in the Hoechst Blue channel rather than in the default forward scatter channel, utilizing the differences in DNA content between Mycoplasma and fragments. Subsequently, we identified and isolated it at single-cell resolution via flow cytometry and successfully sorted infectious Mycoplasma in cell culture. Simultaneously, we validated the accuracy and feasibility of this approach using polymerase chain reaction, fluorescence confocal microscopy, and cryo-electron microscopy. This methodology enabled the diagnosis of Mycoplasma at extremely low concentrations, significantly enhancing the detection efficiency and facilitating the isolation and purification of parasitic Mycoplasma in cell culture instead of pure Mycoplasma culture in artificial media for subsequent studies.

Although spectral flow cytometry has become a ubiquitous tool for cell analysis, the use of spectral cytometry on cell sorters requires additional considerations arising from the unique requirements of sorting workflows. Here, we show that care should be taken when ascertaining the purity of a sort on a spectral cell sorter, as the mismatch of buffers used for initial sample suspension and the buffers used for sort collection can affect the unmixing of the data, potentially giving rise to erroneous purity check results.

Barcoding viable cells combined with pooled sample staining is an effective technique that eliminates batch effects from serial cell staining and facilitates uninterrupted data acquisition. We describe three novel and isotopically pure selenium-containing compounds (SeMals) that are useful cellular labeling tools. The maleimide-functionalized selenophenes (⁷⁶SeMal, ⁷⁷SeMal, and ⁷⁸SeMal) covalently react with cellular sulfhydryl groups and uniquely label cell samples. The SeMal reagents label viable and paraformaldehyde-fixed peripheral blood mononuclear cells (PBMC), are well resolved by the mass cytometer, and have little spill into adjacent channels. They appear non-toxic to viable cells at working concentrations. We used SeMal reagents in combination with four isotopically pure tellurium maleimide reagents (¹²⁴TeMal, ¹²⁶TeMal, ¹²⁸TeMal, and ¹³⁰TeMal) to label 21 individual PBMC samples with unique combinations of selenium and tellurium isotopes (seven donors with three replicates using a 7 isotope pick 2 combinatorial schema). The individually barcoded samples were pooled, stained with an antibody cocktail as a pool, and acquired on the mass cytometer as a single suspension. The single-cell data were de-barcoded into separate sample-specific files after data acquisition, enabling an uninterrupted instrument run. Each donor sample retained its unique phenotypic profile with excellent replicate reproducibility. Unlike current live cell barcoding methods, this approach does not require antibodies to surface markers, allowing for the labeling of all cells regardless of surface antigen expression. Additionally, since selenium and tellurium isotopes are not currently utilized in CyTOF antibody panels, this method expands barcoding options and frees up commonly used isotopes for more detailed cell profiling.