Fluorometric Methods to Measure Bioavailable and Total Heme.

Heme b (iron protoporphyrin IX) is an essential but potentially cytotoxic cofactor, signaling molecule, and nutritional source of iron. Its importance in cell biology and metabolism is underscored by the fact that numerous diseases, including various cancers, neurodegenerative disorders, infectious diseases, anemias, and porphyrias, are associated with the dysregulation of heme synthesis, degradation, trafficking, and/or transport. Consequently, methods to measure, image, and quantify heme in cells are required to better understand the physiology and pathophysiology of heme. Herein, we describe fluorescence-based protocols to probe heme bioavailability and trafficking dynamics using genetically encoded fluorescent heme sensors in combination with various modalities, such as confocal microscopy, flow cytometry, and microplate readers. Additionally, we describe a protocol for measuring total heme and its precursor protoporphyrin IX using a fluorometric assay that exploits porphyrin fluorescence. Together, the methods described enable the monitoring of total and bioavailable heme to study heme homeostatic mechanisms in virtually any cell type and organism.

Purification of Planarian Stem Cells Using a Draq5-Based FACS Approach.

Planarians are flatworms that have the remarkable ability to regenerate entirely new animals. This regenerative ability requires abundant adult stem cells called neoblasts, which are relatively small in size, sensitive to irradiation and the only proliferative cells in the animal. Despite the lack of cell surface markers, fluorescence-activated cell sorting (FACS) protocols have been developed to discriminate and isolate neoblasts, based on DNA content. Here, we describe a protocol that combines staining of far-red DNA dye Draq5, Calcein-AM and DAPI, along with a shortened processing time. This profiling strategy can be used to functionally characterize the neoblast population in pharmacologically-treated or gene knockdown animals. Highly purified neoblasts can be analyzed with downstream assays, such as in situ hybridization and RNA sequencing.

[Chinese expert consensus on the application of flow cytometry for the detectin of circulating plasma cells in plasma cell disorders (2024)].

Flow cytometry plays an important role in the diagnosis and treatment of plasma cell diseases, particularly in the detection of circulating plasma cells (CPCs) in the peripheral blood. A consensus about the normalized use of flow cytometry in detection of CPCs in peripheral blood in clinical practice has been achieved. This consensus is founded on evidence-based principles, which elucidates the timing and value of flow cytometry for the detection of CPCs in the monoclonal gammopathy of undetermined significance, smoldering myeloma, multiple myeloma, and plasma cell leukemia and standardizes flow cytometry in the detection of CPCs in plasma cell diseases.

Rapid Two-Day Baculovirus Titering Using Flow Cytometry.

This chapter outlines a rapid detection method to determine the virus titer of your baculovirus stock. Staining of cells with fluorescently labeled gp64 antibody allows for flow cytometer-based quantitation of baculovirus-infected insect cells. In this assay, Sf9 cells are infected with tenfold serial dilutions of the test virus stock, and baculovirus titers are calculated based on the ratio of infected to uninfected cells 13 to 18 h after inoculation.

In situ Patch-seq analysis of microglia reveals a lack of stress genes as found in FACS-isolated microglia.

We applied the patch-seq technique to harvest transcripts from individual microglial cells from cortex, hippocampus and corpus callosum of acute brain slices from adult mice. After recording membrane currents with the patch-clamp technique, the cytoplasm was collected via the pipette and underwent adapted SMART-seq2 preparation with subsequent sequencing. On average, 4138 genes were detected in 113 cells from hippocampus, corpus callosum and cortex, including microglia markers such as Tmem119, P2ry12 and Siglec-H. Comparing our dataset to previously published single cell mRNA sequencing data from FACS-isolated microglia indicated that two clusters of cells were absent in our patch-seq dataset. Pathway analysis of marker genes in FACS-specific clusters revealed association with microglial activation and stress response. This indicates that under normal conditions microglia in situ lack transcripts associated with a stress-response, and that the microglia-isolation procedure by mechanical dissociation and FACS triggers the expression of genes related to activation and stress.

A Label-Free Optical Flow Cytometry Based-Method for Rapid Assay of Disinfectants' Bactericidal Activity.

Selecting the appropriate disinfectant to control and prevent healthcare-associated infections (HAIs) is a challenging task for environmental health experts due to the large number of available disinfectant products. This study aimed to develop a label-free flow cytometry (FCM) method for the rapid evaluation of bactericidal activity and to compare its efficacy with that of standard qualitative/quantitative suspension tests. The bactericidal efficiency of eight commercial disinfectants containing quaternary ammonium compounds (QACs) was evaluated against four strains recommended by EN 13727 (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus hirae) and four multidrug-resistant pathogens. The proposed FCM protocol measures changes in scattered light and counts following disinfectant exposure, neutralization, and culture steps. Unlike other available FCM-based methods, this approach does not rely on autofluorescence measurements, impedance cytometry, or fluorescent dyes. The FCM scattered light signals revealed both decreased count rates and morphological changes after treatment with minimum inhibitory concentrations (MICs) and higher concentrations for all tested bacteria. The results from the FCM measurements showed excellent correlation with those from standard assays, providing a rapid tool for monitoring the susceptibility profile of clinical, multidrug-resistant pathogens to chemical disinfectants, which could support infection prevention and control procedures for healthcare environments. This label-free FCM protocol offers a novel and rapid tool for environmental health experts, aiding in the optimization of disinfectant selection for the prevention and control of HAIs.

HaloTag display enables quantitative single-particle characterisation and functionalisation of engineered extracellular vesicles.

Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity and other properties. Measuring how incorporation varies across a population of EVs is important for characterising such materials and understanding their function, yet it remains challenging to quantitatively characterise the absolute number of engineered proteins incorporated at single-EV resolution. To address these needs, we developed a HaloTag-based characterisation platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, we employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag-mediated quantification is generally robust across EV analysis methods. We compared HaloTag-labelling to antibody-labelling of EVs using single vesicle flow cytometry, enabling us to measure the substantial degree to which antibody labelling can underestimate proteins present on an EV. Finally, we demonstrate the use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterisation tool which complements and expands existing methods.

Generating and Analyzing High-Parameter Histology Images with Histoflow Cytometry.

The usage of histology to investigate immune cell diversity in tissue sections such as those derived from the central nervous system (CNS) is critically limited by the number of fluorescent parameters that can be imaged at a single time. Most immune cell subsets have been defined using flow cytometry by using complex combinations of protein markers, often requiring four or more parameters to conclusively identify, which is beyond the capabilities of most conventional microscopes. As flow cytometry dissociates tissues and loses spatial information, there is a need for techniques that can retain spatial information while interrogating the roles of complex cell types. These issues are addressed here by creating a method for expanding the number of fluorescent parameters that can be imaged by collecting the signals of spectrally overlapping fluorophores and using spectral unmixing to separate the signals of each individual fluorophore. These images are then processed using an analysis pipeline to take high-parameter histology images and extract single cells from these images so that the unique fluorescent properties of each cell can be analyzed at a single-cell level. Using flow cytometry-like gating strategies, cells can then be profiled into subsets and mapped back onto the histology sections to not only quantify their abundance, but also establish how they interact with the tissue environment. Overall, the simplicity and potential of using histoflow cytometry to study complex immune populations in histology sections is demonstrated.

Performance of immunological assays for universal and differential diagnosis of HTLV-1/2 infection in candidates for blood donations from the Brazilian Amazon.

The present study compares the ability of distinct immunological assays (chemiluminescence immunoassay-CLIA, western blot-WB and flow cytometry-FC-Simplex and Duplex) to detect anti-HTLV (human T-lymphotropic virus) antibodies in candidates for blood donations at the Amazonas State Blood Center (Brazil) between January 2018 and December 2022. Overall, 257,942 samples from candidates for blood donations were screened using CLIA, which led to 0.15% seropositivity for HTLV (409 samples). A total of 151 candidates for blood donations were enrolled for retesting with CLIA followed by additional testing using WB and FC-Simplex and Duplex analysis. Our results demonstrated that 62% (93/151), 20% (30/151) and 17% (26/151) of the samples presented positive results with retesting using CLIA, WB and FC-Simplex analysis, respectively. Additional analysis of the CLIA, WB and FC-Simplex results revealed an overall agreement of 56% for CLIA and WB (22 co-negative; 30 co-positive samples), 48% for CLIA and FC-Simplex (21 co-negative; 24 co-positive samples) and 80% for WB and FC-Simplex (51 co-negative; 23 co-positive samples). Considering the WB as the reference standard for the diagnosis of infection with HTLV-1/2, we observed that the CLIA results of ≤3.0 RLU and >10.0 RLU in the retest can be used define a negative or positive result, respectively, and could be used as new specific cut-off values. The overall agreement between WB and FC-Duplex for accomplishing the differential diagnosis was evaluated and demonstrated 100% correspondence for the diagnosis of HTLV-1 (15/15) and HTLV-2 (7/7). Our findings demonstrate that gaps in the diagnosis of infection with HTLV-1/2 could be overcome by the simultaneous use of distinct immunological assays during retesting of candidates for blood donations.

Ex Vivo Evaluation of the Function of Hematopoietic Stem Cells in Toxicology of Metals.

A variety of metals, e.g., lead (Pb), cadmium (Cd), and lithium (Li), are in the environment and are toxic to humans. Hematopoietic stem cells (HSCs) reside at the apex of hematopoiesis and are capable of generating all kinds of blood cells and self-renew to maintain the HSC pool. HSCs are sensitive to environmental stimuli. Metals may influence the function of HSCs by directly acting on HSCs or indirectly by affecting the surrounding microenvironment for HSCs in the bone marrow (BM) or niche, including cellular and extracellular components. Investigating the impact of direct and/or indirect actions of metals on HSCs contributes to the understanding of immunological and hematopoietic toxicology of metals. Treatment of HSCs with metals ex vivo, and the ensuing HSC transplantation assays, are useful for evaluating the impacts of the direct actions of metals on the function of HSCs. Investigating the mechanisms involved, given the rarity of HSCs, methods that require large numbers of cells are not suitable for signal screening; however, flow cytometry is a useful tool for signal screening HSCs. After targeting signaling pathways, interventions ex vivo and HSCs transplantation are required to confirm the roles of the signaling pathways in regulating the function of HSCs exposed to metals. Here, we describe protocols to evaluate the mechanisms of direct and indirect action of metals on HSCs. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Identify the impact of a metal on the competence of HSCs Basic Protocol 2: Identify the impact of a metal on the lineage bias of HSC differentiation Basic Protocol 3: Screen the potential signaling molecules in HSCs during metal exposure Alternate Protocol 1: Ex vivo treatment with a metal on purified HSCs Alternate Protocol 2: Ex vivo intervention of the signaling pathway regulating the function of HSCs during metal exposure.

Identifying antibiotic-resistant strains via cell sorting and elastic-light-scatter phenotyping.

The proliferation and dissemination of antimicrobial-resistant bacteria is an increasingly global challenge and is attributed mainly to the excessive or improper use of antibiotics. Currently, the gold-standard phenotypic methodology for detecting resistant strains is agar plating, which is a time-consuming process that involves multiple subculturing steps. Genotypic analysis techniques are fast, but they require pure starting samples and cannot differentiate between viable and non-viable organisms. Thus, there is a need to develop a better method to identify and prevent the spread of antimicrobial resistance. This work presents a novel method for detecting and identifying antibiotic-resistant strains by combining a cell sorter for bacterial detection and an elastic-light-scattering method for bacterial classification. The cell sorter was equipped with safety mechanisms for handling pathogenic organisms and enabled precise placement of individual bacteria onto an agar plate. The patterning was performed on an antibiotic-gradient plate, where the growth of colonies in sections with high antibiotic concentrations confirmed the presence of a resistant strain. The antibiotic-gradient plate was also tested with an elastic-light-scattering device where each colony's unique colony scatter pattern was recorded and classified using machine learning for rapid identification of bacteria. Sorting and patterning bacteria on an antibiotic-gradient plate using a cell sorter reduced the number of subculturing steps and allowed direct qualitative binary detection of resistant strains. Elastic-light-scattering technology is a rapid, label-free, and non-destructive method that permits instantaneous classification of pathogenic strains based on the unique bacterial colony scatter pattern. KEY POINTS: • Individual bacteria cells are placed on gradient agar plates by a cell sorter • Laser-light scatter patterns are used to recognize antibiotic-resistant organisms • Scatter patterns formed by colonies correspond to AMR-associated phenotypes.

Noninvasive, label-free image approaches to predict multimodal molecular markers in pluripotency assessment.

Manufacturing regenerative medicine requires continuous monitoring of pluripotent cell culture and quality assessment while eliminating cell destruction and contaminants. In this study, we employed a novel method to monitor the pluripotency of stem cells through image analysis, avoiding the traditionally used invasive procedures. This approach employs machine learning algorithms to analyze stem cell images to predict the expression of pluripotency markers, such as OCT4 and NANOG, without physically interacting with or harming cells. We cultured induced pluripotent stem cells under various conditions to induce different pluripotent states and imaged the cells using bright-field microscopy. Pluripotency states of induced pluripotent stem cells were assessed using invasive methods, including qPCR, immunostaining, flow cytometry, and RNA sequencing. Unsupervised and semi-supervised learning models were applied to evaluate the results and accurately predict the pluripotency of the cells using only image analysis. Our approach directly links images to invasive assessment results, making the analysis of cell labeling and annotation of cells in images by experts dispensable. This core achievement not only contributes for safer and more reliable stem cell research but also opens new avenues for real-time monitoring and quality control in regenerative medicine manufacturing. Our research fills an important gap in the field by providing a viable, noninvasive alternative to traditional invasive methods for assessing pluripotency. This innovation is expected to make a significant contribution to improving regenerative medicine manufacturing because it will enable a more detailed and feasible understanding of cellular status during the manufacturing process.

Protocol for in vitro induction and characterization of murine B cell-induced CD4+ regulatory T cells.

Peyer's patches, splenic, and peritoneal B cells induce regulatory T (Treg-of-B) cells in vitro without exogenous chemicals and cytokines. Treg-of-B cells exert suppressive function both in vitro and in vivo. Here, we demonstrate experimental procedures for the generation and characterization of Treg-of-B cells. We describe steps for isolating B220+ B cells, isolating CD4+CD25- T cells, inducing Treg-of-B cells, identifying Treg-of-B cells by flow cytometry, cytokine analyzing by ELISA, and functional testing by suppression assay. For complete details on the use and execution of this protocol, please refer to Chien et al.1 and Chu et al.2.

SWIFT clustering analysis of intracellular cytokine staining flow cytometry data of the HVTN 105 vaccine trial reveals high frequencies of HIV-specific CD4+ T cell responses and associations with humoral responses.

Introduction: The HVTN 105 vaccine clinical trial tested four combinations of two immunogens - the DNA vaccine DNA-HIV-PT123, and the protein vaccine AIDSVAX B/E. All combinations induced substantial antibody and CD4+ T cell responses in many participants. We have now re-examined the intracellular cytokine staining flow cytometry data using the high-resolution SWIFT clustering algorithm, which is very effective for enumerating rare populations such as antigen-responsive T cells, and also determined correlations between the antibody and T cell responses. Methods: Flow cytometry samples across all the analysis batches were registered using the swiftReg registration tool, which reduces batch variation without compromising biological variation. Registered data were clustered using the SWIFT algorithm, and cluster template competition was used to identify clusters of antigen-responsive T cells and to separate these from constitutive cytokine producing cell clusters. Results: Registration strongly reduced batch variation among batches analyzed across several months. This in-depth clustering analysis identified a greater proportion of responders than the original analysis. A subset of antigen-responsive clusters producing IL-21 was identified. The cytokine patterns in each vaccine group were related to the type of vaccine - protein antigens tended to induce more cells producing IL-2 but not IFN-γ, whereas DNA vaccines tended to induce more IL-2+ IFN-γ+ CD4 T cells. Several significant correlations were identified between specific antibody responses and antigen-responsive T cell clusters. The best correlations were not necessarily observed with the strongest antibody or T cell responses. Conclusion: In the complex HVTN105 dataset, alternative analysis methods increased sensitivity of the detection of antigen-specific T cells; increased the number of identified vaccine responders; identified a small IL-21-producing T cell population; and demonstrated significant correlations between specific T cell populations and serum antibody responses. Multiple analysis strategies may be valuable for extracting the most information from large, complex studies.

A Sample Preparation Procedure for Isobaric Labeling-Based Single-Cell Proteomics.

Mass spectrometry-based proteomics has traditionally been limited by the amount of input material for analysis. Single-cell proteomics has emerged as a challenging discipline due to the ultra-high sensitivity required. Isobaric labeling-based multiplex strategies with a carrier proteome offer an approach to overcome the sensitivity limitations. Following this as the basic strategy, we show here the general workflow for preparing cells for single-cell mass spectrometry-based proteomics. This protocol can also be applied to manually isolated cells when large cells, such as cardiomyocytes, are difficult to isolate properly with conventional fluorescence-activated cell sorting (FACS) sorter methods.

spillR: spillover compensation in mass cytometry data.

MOTIVATION: Channel interference in mass cytometry can cause spillover and may result in miscounting of protein markers. Chevrier et al. introduce an experimental and computational procedure to estimate and compensate for spillover implemented in their R package CATALYST. They assume spillover can be described by a spillover matrix that encodes the ratio between the signal in the unstained spillover receiving and stained spillover emitting channel. They estimate the spillover matrix from experiments with beads. We propose to skip the matrix estimation step and work directly with the full bead distributions. We develop a nonparametric finite mixture model and use the mixture components to estimate the probability of spillover. Spillover correction is often a pre-processing step followed by downstream analyses, and choosing a flexible model reduces the chance of introducing biases that can propagate downstream. RESULTS: We implement our method in an R package spillR using expectation-maximization to fit the mixture model. We test our method on simulated, semi-simulated, and real data from CATALYST. We find that our method compensates low counts accurately, does not introduce negative counts, avoids overcompensating high counts, and preserves correlations between markers that may be biologically meaningful. AVAILABILITY AND IMPLEMENTATION: Our new R package spillR is on bioconductor at bioconductor.org/packages/spillR. All experiments and plots can be reproduced by compiling the R markdown file spillR_paper.Rmd at github.com/ChristofSeiler/spillR_paper.

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages.

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.

The Usefulness of Intraepithelial Lymphocyte Immunophenotype Testing for the Diagnosis of Coeliac Disease in Clinical Practice.

BACKGROUND: The diagnosis of coeliac disease (CD) in adults is based on clinical, serological and histological criteria. The inappropriate performance of intestinal biopsies, non-specificity of mild histological lesions and initiation of a gluten-free diet (GFD) before biopsy may hamper the diagnosis. In these situations, determining the intraepithelial lymphogram of the duodenum by flow cytometry (IEL-FC) can be helpful. OBJECTIVES: To describe the clinical scenarios in which the IEL-FC is used and its impact on the diagnosis of CD. METHODS: All adult patients with suspected CD at three tertiary centres for whom the duodenal histology and IEL-FC were available were identified. Catassi and Fasano's diagnostic criteria and changes to a CD diagnosis after the IEL-FCs were collected. RESULTS: A total of 348 patients were included. The following indications for an IEL-FC formed part of the initial study for CD (38%): negative conventional work-up (32%), already on a GFD before duodenal biopsies (29%) and refractoriness to a GFD (2%). The IEL-FC facilitated a definitive diagnosis in 93% of patients with an uncertain diagnosis who had had a conventional work-up for CD or who were on a GFD before histology. CONCLUSIONS: The IEL-FC facilitates the confirmation or rejection of a diagnosis of CD in clinical scenarios in which a conventional work-up may be insufficient.