Cytometry Part A最新文献

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.

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.

Cationic lipids are widely used for gene delivery. Here, we report the transient formation of nuclear actin filaments in mammalian cells transfected with commercially available transfection reagents regardless of the proteins transfected. Readily detectable with phalloidin, nuclear actin ranges from short filaments to a fully developed network. Nuclear actin filaments persist for hours, peak 20 h after transfection, and may be involved in DNA damage repair.