In Vitro Interaction Between Yeast Extracellular Vesicles and Human Monocyte-Derived Dendritic Cells.

Extracellular vesicles (EVs) are lipid-bound particles produced by a wide variety of cells from different biological species. EVs can carry molecules, such as nucleic acids and metabolites, and are involved in cell functioning, communication, and signaling. Recent literature reported that pathogenic or commensal yeast strains can produce EVs targeting the host's immune system and exerting immunomodulatory actions. In humans, yeast EVs can be endocytosed by dendritic cells (DCs), characterized by phagocyting and migrating capabilities with the role of capturing antigens to present to T lymphocytes, triggering the immune response. Physiological or disease-associated immunosenescence impairs both DC functionality and gut microbiota; thus investigating the interaction between commensal microorganisms and the host's immune system would help elucidate the impact of aging on the immune system-microbiota interplay. We hereby present a protocol for the incubation of in vitro-generated human monocyte-derived DCs with EVs purified from different yeast strains isolated from fermented milk. The protocol includes flow cytometry analysis on DC activation markers and endocytosis assay.

Phenotypic Characterization of B-Lymphocyte Subpopulations in Human Peripheral Blood: A Cost-Effective Seven-Color One-Tube Protocol.

B cells are crucial components of the immune system, responsible for producing specific antibodies in response to infections and vaccines. Despite their uniform appearance, B cells display diverse surface molecules and functional properties, which are not yet fully understood. Apart from antibody production, B cells also play roles in antigen presentation and cytokine secretion, essential for initiating T-cell immune responses. Their significance as disease biomarkers and therapeutic targets has led to increased research focus. However, the lack of standardized protocols for B-cell identification and the variability in defining B-lymphocyte subpopulations pose some challenges. This paper proposes a B-cell identification panel throughout the evaluation of previous cytometry panels and nomenclature heterogeneity for B-cell subpopulations. Major subpopulations recognized in human peripheral blood include transitional, naive, switched memory, unswitched memory, double negative, and plasmablasts, characterized based on their functional and phenotypic features. We present a standardized flow cytometry protocol utilizing surface phenotypic markers (CD3, CD19, IgD, CD27, CD38, and CD24) to differentiate and analyze B-cell subpopulations. This practical and cost-effective panel can be used in various research and laboratory settings. The challenges of standardizing names and markers for classifying B-lymphocyte subpopulations are discussed, along with protocols utilizing multiple markers and gating strategies, allied with the importance of considering viability markers. In summary, this standardized protocol and panel provide a comprehensive approach to identifying B-cell subpopulations to enhance the reproducibility and comparability of B-cell subpopulation studies.

Characterization of Age-Dependent Changes in Skeletal Muscle Repair and Regeneration Using a Mouse Model of Acute Muscle Injury.

Acute skeletal muscle injury initiates a process of necrosis, debris clearance, and ultimately tissue regeneration via myogenesis. While skeletal muscle stem cells (MuSCs) are responsible for populating the proliferative myogenic progenitor pool to fuel muscle repair, recruited and resident immune cells have a central role in the regulation of muscle regeneration via the execution of phagocytosis and release of soluble factors that act directly on MuSCs to regulate myogenic differentiation. Therefore, the timing of MuSC proliferation and differentiation is closely linked to the populations and behaviors of immune cells present within skeletal muscle. This has important implications for aging and muscle repair, as systemic changes in immune system function contribute to a decline in muscle regenerative capacity. Here, we present adapted protocols for the isolation of mononuclear cells from skeletal muscles for the quantification of immune cell populations using flow cytometry. We also describe a cardiotoxin skeletal muscle injury protocol and detail the expected outcomes including immune cell infiltration to the injured sites and formation of new myocytes. As immune cell function is substantially influenced by aging, we extend these approaches and outcomes to aged mice.

Transcription Factor Analysis to Investigate Immunosenescence in Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is linked to various signs of advanced aging, such as premature immunosenescence which occurs due to decline in regenerative ability of T cells. RA T cells develop a unique aggressive inflammatory senescent phenotype with an imbalance of Th17/T regulatory (Treg) cell homeostasis and presence of CD28- T cells. The phenotypic analysis and characterization of T cell subsets become necessary to ascertain if any functional deficiencies exist within with the help of transcription factor (TF) analysis. These subset-specific TFs dictate the functional characteristics of T-cell populations, leading to the production of distinct effector cytokines and functions. Examining the expression, activity, regulation, and genetic sequence of TFs not only aids researchers in determining their importance in disease processes but also aids in immunological monitoring of patients enrolled in clinical trials, particularly in evaluating various T-cell subsets [Th17 (CD3+CD4+IL17+RORγt+) cells and T regulatory (Treg) (CD3+CD4+CD25+CD127-FOXP3+) cells], markers of T-cell aging [aged Th17 cells (CD3+CD4+IL17+RORγt+CD28-), and aged Treg cells (CD3+CD4+CD25+CD127-FOXP3+CD28-)]. In this context, we propose and outline the protocols for assessing the expression of TFs in aged Th17 and Treg cells, highlighting the crucial aspects of this cytometric approach.

γδ T-Cell Immunophenotype for the Study of Human Aging.

Flow cytometry serves as a crucial tool in immunology, allowing for the detailed analysis of immune cell populations. γδ T cells, a subset of T cells, play pivotal roles in immune surveillance and immune aging. Assessing the phenotype and functional capabilities of γδ T cells isolated from whole blood or tissue within the context of human aging yields invaluable insights into the dynamic changes affecting immune function, tissue homeostasis, susceptibility to infections, and inflammatory responses.

Assessment of Viability of Listeria monocytogenes by Flow Cytometry.

In food industry, Listeria monocytogenes contamination can occur accidentally despite the quality control of raw materials and factory. Decontamination processes or inhibitory effects of ingredients/additives in food products are set up to ensure compliance with hygiene and microbiological criteria. These actions represent stresses for the pathogenic agent, causing fluctuations in its physiological states. Moreover, during these environmental stresses, Listeria monocytogenes can enter in a viable but nonculturable (VBNC) state which is not detected by plate counting but by flow cytometry. This technique coupled with cell staining by fluorescent dyes offers the possibility to assess different physiological states based on different cellular parameters: enzymatic activity, transmembrane integrity, membrane potential, and respiratory activity. In this chapter, we present a method to assess the viability of foodborne pathogens using a double-staining principle based on the assessment of membrane integrity and intracellular esterase activity.

Flow cytometry in acute myeloid leukemia and detection of minimal residual disease.

Acute myeloid leukemia (AML) is a common type of acute leukemia (AL), belonging to malignant tumors of the hematopoietic system with the characteristics of rapid disease development, control with extreme difficulties, easy recurrence, poor prognosis, and incidence rate increasing with age. The traditionally diagnostic standard of French American British (FAB), being based on the morphological examination with high human subjectivity, can no longer meet the demand of clinical diagnosis and treatment of AML. Requirements of objective accuracy and low-dose sample, have become the indispensable method for AML diagnosis and monitoring prognosis. Flow cytometry is a modern technology that can quickly and accurately detect the series, antigen distribution, differentiation stage of AML cells, minimal residual lesions after AML therapy, so as to provide the great significance in guiding clinical diagnosis, hierarchical treatment, and prognosis judgement. This article will systematically elaborate on the application of flow cytometry in the diagnosis and classification of AML, and the detection of minimal residual lesions, thereby providing reference significance for dynamic monitoring and prognostic observation of AML with different immune subtypes of FAB.

SCIP: A scalable, reproducible, and open-source pipeline for morphological profiling image cytometry and microscopy data.

Imaging flow cytometry (IFC) provides single-cell imaging data at a high acquisition rate. It is increasingly used in image-based profiling experiments consisting of hundreds of thousands of multi-channel images of cells. Currently available software solutions for processing microscopy data can provide good results in downstream analysis, but are limited in efficiency and scalability, and often ill-adapted to IFC data. In this work, we propose Scalable Cytometry Image Processing (SCIP), a Python software that efficiently processes images from IFC and standard microscopy datasets. We also propose a file format for efficiently storing IFC data. We showcase our contributions on two large-scale microscopy and one IFC datasets, all of which are publicly available. Our results show that SCIP can extract the same kind of information as other tools, in a much shorter time and in a more scalable manner.

Glucose-6-phosphate dehydrogenase deficiency detection using fluorocytometric assay: Evaluation after 1 year of clinical implementation.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common enzymopathy that affects red blood cells (RBCs) and renders them susceptible to oxidative stress. G6PD deficiency can cause hemolytic anemia, especially after exposure to certain drugs or infections. The diagnosis of G6PD deficiency is usually based on spectrophotometric measurement of enzyme activity, but this method has limitations in heterozygous females and in patients with other hematological disorders. In this study, we evaluated the use of flow cytometry as an alternative method for detecting G6PD deficiency in 514 samples (265 females and 249 males) from a clinical laboratory. We compared the results of flow cytometry with those of spectrophotometry and molecular analysis, and assessed the performance of flow cytometry in different subgroups of patients. We found that flow cytometry was able to identify G6PD deficiency in most cases, with high sensitivity and specificity. Flow cytometry also allowed the quantification of the percentage of G6PD-deficient RBCs, which varied among heterozygous females due to X-chromosome inactivation. Moreover, flow cytometry detected several cases of G6PD deficiency that were missed by spectrophotometry, especially in heterozygous females with normal or subnormal enzyme activity. However, flow cytometry also showed some false negative results, mainly in patients with sickle cell disease. Therefore, flow cytometry is a reliable and efficient tool for screening G6PD deficiency, but some precautions should be taken in interpreting the results in patients with other hematological conditions.

Factors to consider before choosing EV labeling method for fluorescence-based techniques.

A well-designed fluorescence-based analysis of extracellular vesicles (EV) can provide insights into the size, morphology, and biological function of EVs, which can be used in medical applications. Fluorescent nanoparticle tracking analysis with appropriate controls can provide reliable data for size and concentration measurements, while nanoscale flow cytometry is the most appropriate tool for characterizing molecular cargoes. Label selection is a crucial element in all fluorescence methods. The most comprehensive data can be obtained if several labeling approaches for a given marker are used, as they would provide complementary information about EV populations and interactions with the cells. In all EV-related experiments, the influence of lipoproteins and protein corona on the results should be considered. By reviewing and considering all the factors affecting EV labeling methods used in fluorescence-based techniques, we can assert that the data will provide as accurate as possible information about true EV biology and offer precise, clinically applicable information for future EV-based diagnostic or therapeutic applications.

Differential lipid signaling from CD4+ and CD8+ T cells contributes to type 1 diabetes development.

Introduction: We reported that Ca2+-independent phospholipase A2ß (iPLA2ß)-derived lipids (iDLs) contribute to type 1 diabetes (T1D) onset. As CD4+ and CD8+ T cells are critical in promoting ß-cell death, we tested the hypothesis that iDL signaling from these cells participates in T1D development. Methods: CD4+ and CD8+ T cells from wild-type non-obese diabetic (NOD) and NOD.iPLA2ß+/- (NOD.HET) mice were administered in different combinations to immunodeficient NOD.scid. Results: In mice receiving only NOD T cells, T1D onset was rapid (5 weeks), incidence 100% by 20 weeks, and islets absent. In contrast, onset was delayed 1 week and incidence reduced 40%-50% in mice receiving combinations that included NOD.HET T cells. Consistently, islets from these non-diabetic mice were devoid of infiltrate and contained insulin-positive ß-cells. Reduced iPLA2ß led to decreased production of proinflammatory lipids from CD4+ T cells including prostaglandins and dihydroxyeicosatrienoic acids (DHETs), products of soluble epoxide hydrolase (sEH), and inhibition of their signaling decreased (by 82%) IFNγ+CD4+ cells abundance. However, only DHETs production was reduced from CD8+ T cells and was accompanied by decreases in sEH and granzyme B. Discussion: These findings suggest that differential select iDL signaling in CD4+ and CD8+ T cells contributes to T1D development, and that therapeutics targeting such signaling might be considered to counter T1D.

Early B lymphocyte subsets in blood predict prognosis in sepsis.

Background: B lymphocytes play a key role in immunosuppression. This study investigated the prognostic value of B cell subsets in sepsis. Methods: Flow cytometry was used to assess peripheral B cell subsets from patients with sepsis on the first and seventh days following admission, as well as 111 healthy controls. The patients were divided into survivors and non-survivors, based on 28-day prognosis. Results: The analysis showed abnormal distribution and selective depletion of B cells and its subsets in the early stages of sepsis. On day 1, compared with survivors, non-survivors showed significant decreases in the proportion and absolute count of transitional (Tr) B cells, reductions in the proportion of CD5+ B cells, and increases in the proportion of double-negative (DN) B cells. On day 7, the proportions and absolute counts of Tr and CD5+ B cells significantly decreased whereas the proportion of DN B cells significantly increased in non-survivors. Ninety-four survivors and 15 non-survivors were included in our paired-sample rank-sum test. Compared to day 1, only the survivors showed significant increases in absolute B, Tr B, and CD5+ B cell counts by day 7. Multivariate Cox regression analysis showed that the proportion of DN B cells on day 1 (hazard ratio = 1.092 [95% confidence interval: 1.035-1.152], P = 0.001) was a risk factor for mortality, and Kaplan-Meier survival curve analysis showed that patients with proportions of DN B cells > 11.81% on day 1 had poorer prognoses. Receiver operating characteristic curve analysis showed that B cell subset parameters could predict mortality (area under the receiver operating characteristic curve [AUC], 0.741) and enhanced the prognostic value of the Acute Physiology and Chronic Health Evaluation II score (AUC, 0.840). Conclusion: Our study revealed that deficiencies of B, Tr B, and CD5+ B cells, as well as a persistent increase in the proportion of DN B cells, were associated with poor prognosis-and that B cell subsets showed predictive value to mortality. These results provide new insights into the roles of B cell subsets in sepsis, as well as ways to better manage its progression and predict its course.

Protocol to characterize mouse dural mast cells by flow cytometry and immunofluorescence.

Mast cells, which constitute tissue-resident immune cells, are distributed in the dural meninges. Here, we provide procedural guidelines for investigating mouse dural mast cells using two techniques. First, we outline the procedures for dural tissue dissection, single-cell isolation, and subsequent surface staining for mast cell identification via flow cytometry. We then describe the techniques employed for whole dura tissue staining to visualize mast cells using confocal and slide scanning microscopy, followed by analysis using Nikon's NIS-Elements Advanced Research software. For complete details on the use and execution of this protocol, please refer to Lin et al.1.

Serotype 15C Streptococcus pneumoniae resistant to classical complement deposition and agglutination by polyclonal rabbit anti-capsular 15B sera.

BACKGROUND: S. pneumoniae (SP) serotypes 15B and 15C are frequent causative agents of invasive pneumococcal disease (IPD), otitis media, and nasopharyngeal colonization in the post PCV13 era. The principal difference between serotypes 15B and 15C is the presence of an O-acetyl group on the pentasaccharide repeating unit of 15B polysaccharide. It remains unclear if antibodies against SP15B polysaccharide demonstrate functional cross reaction with SP15C strains. We compared functional activity of polyclonal rabbit anti-capsular 15B sera against SP15B and SP15C isolates. METHODS: Using flow cytometry we measured complement factors C3c and C4d deposition on SP15B and 15C in the presence of polyclonal rabbit anti capsular 15B sera. We measured the binding of C3c, common to all complement pathways, and C4d, specific to classical pathway, on SP serotypes 15B and 15C when co-incubated with polyclonal rabbit anti capsular 15B sera and antibody depleted complement. Both the proportion of bacteria with complement deposition and the fluorescence intensity were measured. We also measured agglutination as the increase in forward and side scatter. RESULTS: Polyclonal rabbit anti-capsular 15B sera activated classical pathway resulting in deposition of C4d and C3c at high intensity on all SP15B cells but only achieved limited deposition and intensity of C4 with SP15C. Similarly, polyclonal rabbit anti-capsular 15B sera induced agglutination of SP15B strains in a dose dependent manner and limited agglutination of SP15C. CONCLUSIONS: Anti-capsular 15B sera induce limited C4d and C3c deposition, and minimal agglutination with SP15C strains, reflecting lower classical pathway activation in contrast to high C4d and C3c deposition and agglutination of SP15B. These observations support limited functional cross reactivity of anti-15B to SP15C strains and are consistent with the reduction in opsonophagocytic killing of SP15C reported following immunization with vaccines containing 15B polysaccharide.

Phenotype of bovine mononuclear phagocytes- An update.

Studying mononuclear phagocytes by flow cytometry is challenging due to their phenotypic similarities and the high plasticity of monocytic cells. Despite these challenges, significant progress has been made in cattle research through multicolor flow cytometry, transcriptomics of sorted subsets, and single-cell RNA-sequencing. Here, we provide an overview of established and proposed phenotypic classifications in the bovine mononuclear phagocyte system and discuss the challenges of marker discovery.

Ultrasensitive detection of cancer-associated nucleic acids and mutations by primer exchange reaction-based signal amplification and flow cytometry.

The detection of cancer-associated nucleic acids and mutations through liquid biopsy has emerged as a highly promising non-invasive approach for early cancer detection and monitoring. In this study, we report the development of primer exchange reaction (PER) based signal amplification strategy that enables the rapid, sensitive and specific detection of nucleic acids bearing cancer specific single nucleotide mutations using flow cytometry. Using micrometer size beads as support for immobilizing oligonucleotides and programmable PER assembly for target oligonucleotide recognition and fluorescence signal amplification, we demonstrated the versatile detection of target nucleic acids including KRAS oligonucleotide, fragmented mRNAs, and miR-21. Moreover, our detection system can discriminate single base mutations frequently occurred in cancer-associated genes including KRAS, PIK3CA and P53 from cell extracts and circulating tumor DNAs (ctDNAs). The detection is highly sensitive, with a limit of detection down to 27 fM without pre-amplification. In view of a clinical application, we demonstrate the detection of single mutations after extraction and pre-amplification of ctDNAs from the plasma of breast cancer patients. Importantly, our detection strategy enabled the detection of single KRAS mutation even in the presence of 1000-fold excess of wild type (WT) DNA using multi-color flow cytometry detection approach. Overall, our strategy holds immense potential for clinical applications, offering significant improvements for early cancer detection and monitoring.

Real-World Evidence on Prognostic Value of MRD in Multiple Myeloma Using Flow Cytometry.

Minimal residual disease (MRD) is one of the most important prognostic factors in multiple myeloma (MM) and a valid surrogate for progression-free survival (PFS) and overall survival (OS). Recently, MRD negativity was approved as an early clinical endpoint for accelerated drug approval in MM. Nevertheless, there is limited evidence of MRD utility in real-world setting. In this retrospective multicenter study, we report outcomes of 331 newly diagnosed MM patients with MRD evaluation at Day+100 after autologous stem cell transplantation using flow cytometry with a median limit of detection of 0.001%. MRD negativity was reached in 47% of patients and was associated with significantly prolonged median PFS (49.2 months vs. 18.4 months; hazard ratios (HR) = 0.37; p < 0.001) and OS (not reached vs. 74.9 months; HR = 0.50; p = 0.007). Achieving MRD negativity was associated with PFS improvements regardless of age, International Staging System (ISS) stage, lactate dedydrogenase (LDH) level, or cytogenetic risk. Importantly, MRD positive patients benefited from lenalidomide maintenance versus no maintenance (18-months PFS: 81% vs. 46%; HR = 0.24; p = 0.002) while in MRD negative patients such benefit was not observed (p = 0.747). The outcomes of our real-world study recapitulate results from clinical trials including meta-analyses and support the idea that MRD positive patients profit more from lenalidomide maintenance than MRD negative ones.

Single nucleus DNA sequencing of flow sorted archived frozen and formalin fixed paraffin embedded solid tumors.

BACKGROUND: Archived samples, including frozen and formalin fixed paraffin embedded (FFPE) tissues, are a vast resource of clinically annotated materials for the application of high-definition genomics to improve patient management and provide a molecular basis for the delivery of personalized cancer therapeutics. Notably, FFPE tissues are stable, provide repeat sampling of tissues of interest, and can be stored indefinitely at ambient temperature. The development of single cell DNA sequencing (scDNA-seq) technologies provides an unparalleled opportunity for the study of tumor heterogeneity and the identification of often rare subclonal cell populations that drive tumor evolution and progression to advanced therapy resistant disease. However, major limitations to the use of archived tissues for scDNA-seq include the low yields of intact cells in the presence of high levels of subcellular debris in biopsies, and the highly variable quantity and quality of the DNA extracted from samples of interest. The latter is of high significance for the use of FFPE tissues due to the presence of DNA-protein crosslinks. In addition, many samples, notably tumors arising in solid tissues, contain admixtures of reactive stroma, inflammatory cells, and necrosis in immediate contact with tumor cells. RESULTS: To expand their use for translational studies, we optimized flow sorting and sequencing of single nuclei from archived fresh frozen (FF) and FFPE tumor tissues. Our methods, which include isolation of intact nuclei suitable for library preparations, quality control (QC) metrics for each step, and a single cell sequencing bioinformatic processing and analysis pipeline, were validated with flow sorted nuclei from matching FF and FFPE ovarian cancer surgical samples and a sequencing panel of 553 amplicons targeting single nucleotide and copy number variants in genes of interest. CONCLUSIONS: Our flow sorting based protocol provides intact nuclei suitable for snDNA-seq from archival FF and FFPE tissues. Furthermore, we have developed QC steps that optimize the preparation and selection of samples for deep single cell clonal profiling. Our data processing pipeline captures rare subclones in tumors with highly variable genomes based on variants in genes of interest.