Density Gradient Centrifugation-Independent Purification of Human Basophils.

CLL/SLL is the most common leukemia in the western world. The disease is indolent; however, most patients require treatment at some point of the disease course. Outside of allogeneic transplants, the treatment is rarely curative but often controls CLL/SLL manifestations for many years. Several lines of therapy may be used sequentially to prolong clinical remission. Because of the prolonged disease course, CLL/SLL monitoring represents a sizable portion of the workload in a typical flow cytometry laboratory involved in the diagnosis and monitoring of hematopoietic neoplasms. Minimal/measurable disease monitoring of CLL/SLL has emerged as a key component in treatment monitoring and sequencing. In the face of effective therapies, clinical laboratories are tasked with monitoring ever smaller proportions of MRD with high precision and accuracy. With the recent addition of surface antigen-targeting biologics such as antibodies and CAR-T cells, the task has become more complex due to the unavailability of commonly analyzed antigens for flow cytometric analysis. This article details a flow cytometric test developed at Memorial Sloan Kettering Cancer Center that has proven to consistently achieve high sensitivity (<0.01% of nucleated cells) in the bone marrow and peripheral blood, even when CD19 is lost or unavailable for analysis. Moreover, the test helps distinguish between CLL and other CD5-positive B cell neoplasms. The Basic Protocol provides a detailed operational procedure for processing, staining, and cytometric acquisition of samples. The Support Protocol provides typical steps and caveats for MRD data analysis in CLL/SLL and in distinguishing CLL/SLL from other B cell neoplasms and normal CD5-positive B cells. © 2022 Wiley Periodicals LLC. Basic Protocol: Processing, staining, and cytometric analysis of bone marrow or peripheral blood cells for MRD analysis of CLL/SLL Support Protocol: Analysis and interpretation of CLL MRD assay.

SPAdes-St. Petersburg genome Assembler-was originally developed for de novo assembly of genome sequencing data produced for cultivated microbial isolates and for single-cell genomic DNA sequencing. With time, the functionality of SPAdes was extended to enable assembly of IonTorrent data, as well as hybrid assembly from short and long reads (PacBio and Oxford Nanopore). In this article we present protocols for five different assembly pipelines that comprise the SPAdes package and that are used for assembly of metagenomes and transcriptomes as well as assembly of putative plasmids and biosynthetic gene clusters from whole-genome sequencing and metagenomic datasets. In addition, we present guidelines for understanding results with use cases for each pipeline, and several additional support protocols that help in using SPAdes properly. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Assembling isolate bacterial datasets Basic Protocol 2: Assembling metagenomic datasets Basic Protocol 3: Assembling sets of putative plasmids Basic Protocol 4: Assembling transcriptomes Basic Protocol 5: Assembling putative biosynthetic gene clusters Support Protocol 1: Installing SPAdes Support Protocol 2: Providing input via command line Support Protocol 3: Providing input data via YAML format Support Protocol 4: Restarting previous run Support Protocol 5: Determining strand-specificity of RNA-seq data.

Spatial transcriptomic technologies have been developed rapidly in recent years. The addition of spatial context to expression data holds the potential to revolutionize many fields in biology. However, the lack of computational tools remains a bottleneck that is preventing the broader utilization of these technologies. Recently, we have developed Giotto as a comprehensive, generally applicable, and user-friendly toolbox for spatial transcriptomic data analysis and visualization. Giotto implements a rich set of algorithms to enable robust spatial data analysis. To help users get familiar with the Giotto environment and apply it effectively in analyzing new datasets, we will describe the detailed protocols for applying Giotto without any advanced programming skills. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Getting Giotto set up for use Basic Protocol 2: Pre-processing Basic Protocol 3: Clustering and cell-type identification Basic Protocol 4: Cell-type enrichment and deconvolution analyses Basic Protocol 5: Spatial structure analysis tools Basic Protocol 6: Spatial domain detection by using a hidden Markov random field model Support Protocol 1: Spatial proximity-associated cell-cell interactions Support Protocol 2: Assembly of a registered 3D Giotto object from 2D slices.

Cell‐cell interactions are required for development and homeostasis in multicellular organisms from insects to mammals. A critical process governed by these interactions is cell competition, which functions throughout development to control tissue composition by eliminating cells that possess a lower fitness status than their neighbors. Human pluripotent stem cells (hPSCs) are a key biological tool in modeling human development and offer further potential as a source of clinically relevant cell populations for regenerative medicine applications. Recently, cell competition has been demonstrated in hPSC cultures and during induced pluripotent stem cell reprogramming. In turn, these findings suggest that hPSCs can be used as a tool to study and model cell‐cell interactions during different stages of development and disease. Here, we provide a panel of protocols optimized for hPSCs to investigate the potential role that cell competition may have in determining the fate and composition of cell populations during culture. The protocols entail assessment of the competitive phenotype and the mode through which cell competition may lead to elimination of less‐fit cells from mosaic cultures with fitter counterparts. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Electroporation of hPSCs to establish a fluorescent reference cell line Support Protocol 1: Single‐cell dissociation of hPSCs Support Protocol 2: Single‐cell cloning of fluorescently labeled hPSCs Basic Protocol 2: Separate culture and co‐culture proliferation assays Basic Protocol 3: Assessing levels of apoptosis in hPSC cultures using flow cytometry Basic Protocol 4: Transwell assay Support Protocol 3: Immunohistochemistry and image quantification of cleaved caspase‐3 Basic Protocol 5: Cell confrontation assay Basic Protocol 6: Cell compression assay Basic Protocol 7: Time‐lapse imaging to assess mechanical extrusion

Entamoeba histolytica is a parasitic protozoan and the causative agent of amoebiasis in humans. Amoebiasis has a high incidence of disease, resulting in ∼67,900 deaths per year, and it poses a tremendous burden of morbidity and mortality in children. Despite its importance, E. histolytica is an understudied parasite. These protocols describe the in vitro growth, maintenance, cryopreservation, genetic manipulation, and cloning of axenic E. histolytica trophozoites. There has been significant progress in genetic manipulation of this organism over the past decade, and these protocols outline the ways in which these advances can be implemented. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Culturing E. histolytica trophozoites Support Protocol 1: Preparation of TYI-S-33 medium Support Protocol 2: Lot testing of Biosate peptone and adult bovine serum for TYI-S-33 medium Basic Protocol 2: Cryopreservation of E. histolytica trophozoites Support Protocol 3: Preparation of cryoprotectant solutions Basic Protocol 3: Transfection of E. histolytica trophozoites with Attractene reagent Basic Protocol 4: Creating clonal lines using limiting dilution Basic Protocol 5: Knockdown of one to two genes with trigger-induced RNA interference Support Protocol 4: Evaluation of RNA interference knockdown with reverse transcriptase PCR Basic Protocol 6: E. histolytica growth curves.

Immunoheterogeneity is a potential hindrance to the maximum applicability of mesenchymal stromal cells (MSCs) in biotherapeutics and regenerative medicine. The primary causes of this heterogeneityare donor variation, diversity of tissue sources, and the culture conditionsunder which MSCs clonally propagate. Immunoheterogeneity could manifest as functional heterogeneity within these stem cells, affecting their ability to differentiate into adult tissue lineages. The impacted healthy human third molar tooth is a rich source of MSCs, which are isolatable both from the dental pulp and the dental follicle. Dental pulp stem cells (DPSCs), though widely characterized for their multipotential lineage differentiation, demonstrate considerable immunoheterogeneity, which demands the enrichment of these stem cells post-isolation. The aim of this study is to enrich and understand the functional differences in sub-populations of MSCs towards a specific lineage. In this protocol, we have highlighted detailed, stepwise methods for sorting and culturing post-sort and optimized methods for quantifying the differentiation of sorted DPSCs towards the osteogenic lineage. Our workflow is designed with additional considerations for cell sorter availability, maintenance of cell viability during long-distance transport to a state-of-the-art facility, and optimization of post-sorting cell culture and downstream experiments. This protocol can be universally followed to understand functional differences within MSCs isolated from different tissue sources and their implications for multi-lineage differentiation. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Assessing multiparametric expression of MSC surface markers and single cell sorting for enrichment of stem cells Support Protocol 1: Antibody panel design Support Protocol 2: MSC culture, preparation of cell suspension, and sample staining Support Protocol 3: Setting up controls Support Protocol 4: Analysis and data interpretation Basic Protocol 2: Quantitative and qualitative assessment of osteogenic differentiation of post-sorted DPSCs Support Protocol 5: Differentiation of MSCS (pre-sort and post-sort considerations) Support Protocol 6: Cytochemical staining and quantification Support Protocol 7: Immunofluorescence staining Support Protocol 8: RNA isolation and qPCR-based characterization.

Mucopolysaccharidoses (MPSs) are complex lysosomal diseases that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, and tissues. Lysosomal enzymes responsible for GAG degradation are defective in MPSs. GAGs including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) are biomarkers for MPSs. This article describes a stable isotope dilution-tandem mass spectrometric method for quantifying CS, DS, and HS in urine samples. The GAGs are methanolyzed to uronic/iduronic acid-N-acetylhexosamine or uronic/iduronic acid-N-glucosamine dimers and mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from HS, DS, and CS are separated by ultra-performance liquid chromatography (UPLC) and analyzed by electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. This UPLC-MS/MS GAG assay is useful for identifying patients with MPS types I, II, III, VI, and VII. © 2025 Wiley Periodicals LLC. Basic Protocol: Urinary GAG analysis by ESI-MS/MS Support Protocol 1: Prepare calibration samples Support Protocol 2: Preparation of stable-isotope-labeled internal standards Support Protocol 3: Preparation of quality controls for GAG analysis in urine Support Protocol 4: Optimization of methanolysis time Support Protocol 5: Measurement of methanolic HCl concentration Support Protocol 6: Preparation of working methanolic HCl solution (1.1 M) Support Protocol 7: Dilution of prepared urine sample.

Allergen challenge in allergic rhinitis rapidly induces increased peripheral blood type 2 innate lymphoid cells that express CD84

Our understanding of programmed cell death 1 (PD-1) biology is limited due to technical difficulties in establishing reproducible, yet simple, in vitro assays to study PD-1 signaling in primary human T cells. The protocols in this article were refined to test the consequences of PD-1 ligation on short-term T cell signaling, long-term T cell function, and the structural consequences of PD-1 ligation with PD-1 ligands. Basic Protocol 1 addresses the need for a robust and reproducible short-term assay to examine the signaling cascade triggered by PD-1. We describe a phospho flow cytometry method to determine how PD-1 ligation alters the level of CD3ζ phosphorylation on Tyr142 , which can be easily applied to other proximal signaling proteins. Basic Protocol 2 describes a plate-bound assay that is useful to examine the long-term consequences of PD-1 ligation such as cytokine production and T cell proliferation. Complementary to that, Basic Protocol 3 describes an in vitro superantigen-based assay to evaluate T cell responses to therapeutic agents targeting the PD-1/PD-L axis, as well as immune synapse formation in the presence of PD-1 engagement. Finally, in Basic Protocol 4 we outline a tetramer-based method useful to interrogate the quality of PD-1/PD-L interactions. These protocols can be easily adapted for mouse studies and other inhibitory receptors. They provide a valuable resource to investigate PD-1 signaling in T cells and the functional consequences of various PD-1-based therapeutics on T cell responses. © 2020 Wiley Periodicals LLC. Basic Protocol 1: PD-1 crosslinking assay to determine CD3ζ phosphorylation in primary human T cells Basic Protocol 2: Plate-based ligand binding assay to study PD-1 function in human T cells Support Protocol 1: T cell proliferation assay in the presence of PD-1 ligation Basic Protocol 3: In vitro APC/T cell co-culture system to evaluate therapeutic interventions targeting the PD-1/PD-L1 axis Support Protocol 2: Microscopy-based approach to evaluate the consequences of PD-1 ligation on immune synapse formation Basic Protocol 4: Tetramer-based approach to study PD-1/PD-L1 interactions.

Basophil Activation Test Discriminates Between Allergy and Tolerance Among Peanut Sensitized Children

Preface

Three-dimensional (3D) cerebral cortical organoids are popular in vitro cellular model systems widely used to study human brain development and disease, compared to traditional stem cell-derived methods that use two-dimensional (2D) monolayer cultures. Despite the advancements made in protocol development for cerebral cortical organoid derivation over the past decade, limitations due to biological, mechanistic, and technical variables remain in generating these complex 3D cellular systems. Building from our previously established differentiation system, we have made modifications to our existing 3D cerebral cortical organoid protocol that resolve several of these technical and biological challenges when working with diverse groups of human induced pluripotent stem cell (hiPSC) lines. This improved protocol blends a 2D monolayer culture format for the specification of neural stem cells and expansion of neuroepithelial progenitor cells with a 3D system for improved self-aggregation and subsequent organoid development. Furthermore, this "hybrid" approach is amenable to both an accelerated cerebral cortical organoid protocol as well as an alternative long-term differentiation protocol. In addition to establishing a hybrid technical format, this protocol also offers phenotypic and morphological characterization of stage-specific cellular profiles using antibodies and fluorescent-based dyes for live cell imaging. © 2024 Wiley Periodicals LLC. Basic Protocol 1: hiPSC-based 2D monolayer specification into neural stem cells (NSCs) Basic Protocol 2: Serial passaging and 2D monolayer expansion of neuroepithelial progenitor cells (NPCs) Support Protocol 1: Direct cryopreservation and rapid thawing of NSCs and NPCs Basic Protocol 3: Bulk aggregation of 3D neurospheres and accelerated cerebral cortical organoid differentiation Alternate Protocol 1: Bulk aggregation of 3D neurospheres and long-term cerebral cortical organoid differentiation Support Protocol 2: High-throughput 3D neurosphere formation and 2D neurosphere migration assay Support Protocol 3: LIVE/DEAD stain cell imaging assay of 3D neurospheres Support Protocol 4: NeuroFluor NeuO live cell dye for 3D cerebral cortical organoids.

For the diagnosis of allergy, cellular basophil activation tests (BAT), e.g. histamine or sulfidoleukotriene release tests, have long been introduced, but the expression of basophil activation markers such as CD63 and CD203c detected by flow cytometry has attracted more recent attention. A recent opinion paper in this Journal has stressed not only the potential but also the possible pitfalls of flow-cytometric BAT. We have applied clinical validation of various BAT in various ways for several years, and our experience shows that these new technologies have more potentials and perspectives than pitfalls. A comprehensive review of clinically validated studies on allergy to aeroallergens, insect venoms, latex, food allergens and drugs, e.g. myorelaxants, β-lactams, pyrazolones and non-steroidal anti-inflammatory drugs, as well as chronic urticaria shows clearly that even with different protocols, reproducible and meaningful results can be obtained. Although the available technologies may still be optimized and better standardized, there are no serious reasons to deprive allergic patients of clinically indicated BAT, which can be performed reliably by any laboratory with allergy and flow-cytometric capacity and expertise.

Background. Chronic spontaneous urticaria (CSU) is a condition with heterogeneous immunopathogenic mechanisms. Identifying the autoimmune type of CSU is necessary for choosing proper therapeutic tactics. Objective. To evaluate the use of basophil activation test in clinical practice to diagnose the autoimmune type of CSU. Materials and methods. The study included 125 patients with CSU aged 18 to 75 years (median 42 years); 72.8% were females. All patients were evaluated for urticaria activity using the UAS7 score and thyroid function; eosinophil and basophil counts and total IgE were determined in peripheral blood, an intradermal test with autologous serum test and an indirect basophil activation test (BAT) was performed using flow cytometry. Results. A positive BAT result was obtained in 43 (34.4%) patients with CSU. There was a higher activity of urticaria in BAT(+) patients vs. BAT(–) ones (p=0.031), angioedema (p=0.012), and hypothyroidism (p=0.002) were also more common in BAT(+) patients. There were lower eosinophil (p=0.027) and basophil (p=0.026) counts and total blood IgE (p&lt;0.001), and a higher level of antibodies to thyroid peroxidase (p=0.004). Conclusion. The inclusion of indirect BAT in the algorithm of examination of patients with CSU enables identifying cases with an autoimmune type of disease in clinical practice.

COVID-19, caused by SARS-CoV-2, has had a significant global impact. While vaccines and treatments have reduced severe cases and deaths, the long-term effects are not yet well understood. Current models used for research, such as non-human primates and transgenic mice, are expensive and require scarce Biosafety Level-3 (BSL-3) laboratories, thereby limiting their practicality. However, the mouse hepatitis virus 1 (MHV-1) mouse model offers a promising alternative. This surrogate model can be investigated in more widely available Biosafety Level-2 (BSL-2) laboratories. Furthermore, mice are affordable and easy to handle, and utilizing MHV-1 as a surrogate for SARS-CoV-2 eliminates the need for costly transgenic mice. Importantly, the MHV-1 model successfully recapitulates COVID-19-related clinical symptoms, weight loss, multiorgan pathological changes and failure in acute stages, irreversible neurological complications, and other long-term organ dysfunction post-infection, which are similar to available human data post-COVID-19. To assist researchers in establishing and using the MHV-1 mouse model, this protocol offers comprehensive guidance encompassing procedures for animal preparation, induction of viral infection, clinical observation, pathological changes, and tissue analysis for mechanistic studies, thereby yielding valuable insights into disease mechanisms and progression. By adopting the MHV-1 model and the provided protocols, researchers can effectively circumvent financial constraints and the limited availability of BSL-3 laboratories, thus facilitating a more accessible and cost-effective approach to investigating the underlying mechanisms of SARS-CoV-2 pathophysiology and exploring potential therapeutic interventions. © 2023 Wiley Periodicals LLC. Basic Protocol: Induction of mouse hepatitis virus 1 (MHV-1) infection in A/J mice Support Protocol 1: Histological evaluation Support Protocol 2: Liver enzyme measurement Support Protocol 3: Western blot analysis of aquaporin expression Support Protocol 4: mRNA measurement Support Protocol 5: Immunohistochemistry/immunofluorescence Support Protocol 6: Tissue water measurement.