Search results for "Mass cytometry"
showing 8 items of 8 documents
Deep phenotypical characterization of human CD3\(^{+}\)CD56\(^{+}\) T cells by mass cytometry
2020
CD56\(^{+}\) T cells are a group of pro‐inflammatory CD3\(^{+}\) lymphocytes with characteristics of natural killer cells, being involved in antimicrobial immune defense. Here, we performed deep phenotypic profiling of CD3\(^{+}\)CD56\(^{+}\) cells in peripheral blood of normal human donors and individuals sensitized to birch‐pollen or/and house dust mite by high‐dimensional mass cytometry combined with manual and computational data analysis. A co‐regulation between major conventional T‐cell subsets and their respective CD3\(^{+}\)CD56\(^{+}\) cell counterparts appeared restricted to CD8\(^{+}\), MAIT, and TCRγδ\(^{+}\) T‐cell compartments. Interestingly, we find a co‐regulation of several …
Guidelines for the use of flow cytometry and cell sorting in immunological studies
2017
The marriage between immunology and cytometry is one of the most stable and productive in the recent history of science. A rapid search in PubMed shows that, as of July 2017, using “flow cytometry immunology” as a search term yields more than 68 000 articles, the first of which, interestingly, is not about lymphocytes. It might be stated that, after a short engagement, the exchange of the wedding rings between immunology and cytometry officially occurred when the idea to link fluorochromes to monoclonal antibodies came about. After this, recognizing different types of cells became relatively easy and feasible not only by using a simple fluorescence microscope, but also by a complex and some…
Heterogeneity of circulating CD8 T-cells specific to islet, neo-antigen and virus in patients with type 1 diabetes mellitus
2018
Auto-reactive CD8 T-cells play an important role in the destruction of pancreatic β-cells resulting in type 1 diabetes (T1D). However, the phenotype of these auto-reactive cytolytic CD8 T-cells has not yet been extensively described. We used high-dimensional mass cytometry to phenotype autoantigen- (pre-proinsulin), neoantigen- (insulin-DRIP) and virus- (cytomegalovirus) reactive CD8 T-cells in peripheral blood mononuclear cells (PBMCs) of T1D patients. A panel of 33 monoclonal antibodies was designed to further characterise these cells at the single-cell level. HLA-A2 class I tetramers were used for the detection of antigen-specific CD8 T-cells. Using a novel Hierarchical Stochastic Neighb…
Cell population analysis of the adult murine subependymal neurogenic lineage by flow cytometry
2021
Summary This protocol provides a flow-cytometry-based procedure to classify and isolate all cells of the adult rodent subependymal zone (SEZ) neurogenic lineage, without the need for reporter mice, into different cell populations, including three neural stem cell (NSC) fractions with molecular signatures that are coherent with single-cell transcriptomics. Additionally, their cycling behavior can be assessed by means of 5-ethynyl-2′-deoxyuridine (EdU) incorporation. Our method allows the isolation of different NSC fractions and the functional assay of their cycling heterogeneity and quiescence-activation transitions. For complete details on the use, execution, and outcomes of this protocol, …
High Dimensional Immune Profiling Reveals Different Response Patterns in Active and Latent Tuberculosis Following Stimulation With Mycobacterial Glyc…
2021
Upon infection withMycobacterium tuberculosis(Mtb) the host immune response might clear the bacteria, control its growth leading to latent tuberculosis (LTB), or fail to control its growth resulting in active TB (ATB). There is however no clear understanding of the features underlying a more or less effective response. Mtb glycolipids are abundant in the bacterial cell envelope and modulate the immune response to Mtb, but the patterns of response to glycolipids are still underexplored. To identify the CD45+leukocyte activation landscape induced by Mtb glycolipids in peripheral blood of ATB and LTB, we performed a detailed assessment of the immune response of PBMCs to the Mtb glycolipids lip…
FACS-based protocol to assess cytotoxicity and clonogenic potential of colorectal cancer stem cells using a Wnt/β-catenin signaling pathway reporter
2021
Summary Cancer stem cells (CSCs) play a key role in tumor initiation and progression. A real-time tool to evaluate the activation of CSC-specific signaling pathways is crucial for the study of this cancer cell subset. Here, we present a protocol to monitor, in vitro, the activation of Wnt/β-catenin signaling pathway, which is considered a functional biomarker for colorectal CSCs (CR-CSCs). This flow-cytometry-based protocol allows it to isolate CR-CSCs and to evaluate their cytotoxicity upon anti-tumor treatments. For complete details on the use and execution of this protocol, please refer to Di Franco et al. (2021).
Characterisation of gd T cells infiltrating colorectal cancer
2021
We have read with great interest the paper by de Vries et al 1 reporting on the immune landscape of colorectal cancer (CRC) by high-dimensional mass cytometry, flow cytometry and single cell RNA sequencing. Among clusters of immune cells infiltrating CRC, authors have identified two populations of γδ T cells: one programmed cell death protein 1 (PD-1)+ population is almost exclusively found in DNA mismatch repair (MMR)-deficient (d) tumours, constitutes up to 8.4% of CD45+ cells and has an activated phenotype, and a PD-1− counterpart with a resting phenotype, which is also found in colorectal normal mucosa and MMR-proficient (p) tumours. Using deconvolution of transcriptomic datasets and si…
Reduced CD27−IgD− B Cells in Blood and Raised CD27−IgD− B Cells in Gut-Associated Lymphoid Tissue in Inflammatory Bowel Disease
2019
The intestinal mucosa in inflammatory bowel disease (IBD) contains increased frequencies of lymphocytes and a disproportionate increase in plasma cells secreting immunoglobulin (Ig)G relative to other isotypes compared to healthy controls. Despite consistent evidence of B lineage cells in the mucosa in IBD, little is known of B cell recruitment to the gut in IBD. Here we analyzed B cells in blood of patients with Crohn's disease (CD) and ulcerative colitis (UC) with a range of disease activities. We analyzed the frequencies of known B cell subsets in blood and observed a consistent reduction in the proportion of CD27−IgD− B cells expressing all Ig isotypes in the blood in IBD (independent o…