Cells Basic Protocol Research Articles

Efficient CRISPR/Cas9 Knock-in Approaches for Manipulation of Endogenous Genes in Human B Lymphoma Cells.

Precise understanding of temporally controlled protein-protein interactions, localization, and expression is often difficult to achieve using traditional overexpression techniques. Recent advances have made CRISPR-based knock-in approaches efficient, which enables rapid derivation of cells with tagged endogenous proteins. However, the high degree of variability in knock-in efficiency across cell types and gene loci poses challenges, in particular with B lymphocytes, which are refractory to lipid transfection. Here, we present detailed protocols for efficient B lymphoma cell CRISPR/Cas9-mediated knock-in. We address knock-in efficiency in two ways. First, we provide a detailed approach for assessing cutting efficiency to select the most efficient single guide RNA for the gene region of interest. Second, we provide detailed approaches for tagging endogenous proteins with a fluorescent marker or instead for co-expressing them with an unlinked fluorescent marker. Either approach facilitates downstream selection of single-cell or bulk populations with the desired knock-in, particularly when knock-in efficiency is low. The utility of this approach is demonstrated via examples of engineering tags onto endogenous protein N- or C-termini, together with downstream analyses. We anticipate that this workflow can be applied more broadly to other cell types for efficient knock-in into endogenous loci. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Choosing an optimal knock-in target site and single guide RNA (sgRNA) design Basic Protocol 2: Assessment of Cas9 editing efficiency at the desired B cell genomic knock-in site Basic Protocol 3: Cloning the sgRNA dual guide construct Basic Protocol 4: Repair template design and cloning Basic Protocol 5: Electroporation and selection of engineered B cells Basic Protocol 6: Single-cell cloning of engineered B cells.

Optimizing CAR-NK Cell Transduction and Expansion: Leveraging Cytokine Modulation for Enhanced Performance.

Cellular immunotherapy has emerged as one of the most potent approaches to treating cancer patients. Adoptive transfer of chimeric antigen receptor (CAR) T cells as well as the use of haploidentical natural killer (NK) cells can induce remission in patients with lymphoma and leukemia. Although the use of CAR T cells has been established, this approach is currently limited for wider use by the risk of severe adverse events, including cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. Moreover, the risk of triggering graft vs host reactions in settings of allogeneic T cell infusion limits the use to autologous CAR T cells if advanced CRISPR engineering is not applied. In contrast, NK cell-based cancer immunotherapy has emerged as a safe approach even in allogeneic settings. However, efficient transduction of primary blood NK cells with vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentivirus commonly used for T cell modification remains challenging. This article presents a detailed method that significantly enhances the transduction efficiency of NK cells by utilizing a short-term culture in cytokine-supplemented medium. It also encompasses the preparation of high-titer and high-quality lentiviral particles for optimal NK cell transduction. Overall, this protocol details the step-by-step culture of NK cells in cytokine-supplemented medium, their transduction with VSV-G lentiviral vectors, and subsequent expansion for functional assays. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of NK cells from human peripheral blood mononuclear cells (PBMCs) Basic Protocol 2: NK cell expansion and transduction with lentivirus for generating CAR-NK cells Support Protocol 1: Plasmid amplification Support Protocol 2: Lentivirus preparation Support Protocol 3: Lentivirus titration.

A Hybrid 2D-to-3D in vitro Differentiation Platform Improves Outcomes of Cerebral Cortical Organoid Generation in hiPSCs.

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.

Set Up and Utilization of a Three-Dimensional In Vitro Bioreactor System for Human Intestinal Studies and Microbial Co-Cultures.

The study of human intestinal physiology and host-microbe interactions is crucial for understanding gastrointestinal health and disease. Traditional two-dimensional cell culture models lack the complexity of the native intestinal environment, limiting their utility in studying intestinal biology. Here, we present a detailed protocol for the set up and utilization of a three-dimensional (3D) in vitro bioreactor system for human intestinal studies and bacterial co-culture. This article outlines the design and assembly of the bioreactor system, scaffold fabrication, bacterial culture techniques, analysis methods, and troubleshooting tips. By providing step-by-step instructions, the goal is to enable other laboratories to utilize physiologically relevant tissue models of the human intestine, incorporating key features, such as nutrient flow, multiple human cell types, 3D architecture, and microbial communities. The incorporation of commensal bacteria into the bioreactor system allows for the investigation of complex host-microbe interactions, providing insight into gastrointestinal health and pathology. This article serves as a comprehensive resource for scientists seeking to advance their understanding of intestinal biology toward the development of novel therapeutic strategies for gastrointestinal disorders. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Scaffold design Basic Protocol 2: Intestinal cell culture: Caco2 cells Basic Protocol 3: Intestinal cell culture: organoids Basic Protocol 4: Bioreactor design and set up Basic Protocol 5: Bacteria in 3D bioreactor set up Basic Protocol 6: Bacteria and drug dosing.

Concanavalin-A-Induced Acute Liver Injury in Mice.

Acute liver injury is a life-threatening disease. Although immune responses are involved in the development and exacerbation of acute liver injury, the cellular and molecular mechanisms are not fully understood. Intravenous administration of the plant lectin concanavalin A (ConA) is widely used as a model of acute liver injury. ConA triggers T cell activation and cytokine production by crosslinking glycoproteins, including the T cell receptor, leading to the infiltration of myeloid cells into the liver and the subsequent amplification of inflammation in the liver. Thus, the pathogenesis of ConA-induced acute liver injury is considered a model of immune-mediated acute liver injury or autoimmune hepatitis in humans. However, the severity of the liver injury and the analyses of immune cells and non-hematopoietic cells in the liver following ConA injection are significantly influenced by the experimental conditions. This article outlines protocols for ConA-induced acute liver injury in mice and evaluation methods for liver injury, immune cells, and non-hematopoietic cells in the liver. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Induction of acute liver injury by ConA injection Basic Protocol 2: Evaluation of inflammatory cytokines in mouse plasma Basic Protocol 3: Preparation of liver sections and histological analysis of liver injury Basic Protocol 4: Preparation of liver immune cells Basic Protocol 5: Preparation of hepatocytes, endothelial cells, and hepatic stellate cells Basic Protocol 6: Flow cytometry of immune and non-hematopoietic liver cells Basic Protocol 7: Flow cytometric sorting of endothelial cells and hepatic stellate cells Basic Protocol 8: Quantitative reverse transcription polymerase chain reaction.

Generation of Murine Chimeric Antigen Receptor-Modified T Cells for In Vivo Studies in Syngeneic Tumor Models.

CAR-T cell therapy has emerged as a potent and effective tool in the immunotherapy of refractory cancers. However, challenges exist in their clinical application, necessitating extensive preclinical research to optimize their function. Various preclinical in vitro and in vivo models have been proposed for such purpose; among which immunocompetent mouse models serve as an invaluable tool in studying host immune interactions within a more realistic simulation of the tumor milieu. We hereby describe a standardized protocol for the generation of high-titer γ-retroviral vectors through transfection of the HEK293T packaging cell line. The virus-containing supernatant is further concentrated using an inhouse concentrator solution, titrated, and applied to mouse T cells purified via a convenient and rapid method by nylon-wool columns. Using the method presented here, we were able to achieve high titer γ-retrovirus and highly pure mouse T cells with desirable CAR transduction efficiency. The mouse CAR T cells produced through this protocol demonstrate favorable CAR expression and viability, thus making them suitable for further in vitro/in vivo assays. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Production of γ-retroviral vectors from retrovirus-backbone plasmids Basic Protocol 2: Concentration of γ-retrovirus-containing supernatants Basic Protocol 3: Titration of concentrated γ-retrovirus Basic Protocol 4: Isolation and activation of mouse T cells Basic Protocol 5: Transduction of activated mouse T cells, assessment of CAR expression, and expansion of CAR T cells for further in vitro/in vivo studies Support Protocol: Surface staining of cells for flow cytometric assessment of CAR expression.

Expression and Purification of Human U1-70K (snRNP70) and its BAD Domains Using an E. coli Expression System.

U1-70K (snRNP70) serves as an indispensable protein component within the U1 complex, assuming a pivotal role in both constitutive and alternative RNA splicing processes. Notably, U1-70K engages in interactions with SR proteins, instigating the assembly of the spliceosome. This protein undergoes regulation through phosphorylation at multiple sites. Of significant interest, U1-70K has been implicated in Alzheimer's disease, in which it tends to form detergent-insoluble aggregates. Even though it was identified more than three decades ago, our understanding of U1-70K remains notably constrained, primarily due to challenges such as low levels of recombinant expression, susceptibility to protein degradation, and insolubility. In endeavoring to address these limitations, we devised a multifaceted approach encompassing codon optimization, strategic purification, and a solubilization protocol. This methodology has enabled us to achieve a high yield of full-length, soluble U1-70K, paving the way for its comprehensive biophysical and biochemical characterization. Furthermore, we provide a detailed protocol for the preparation of phosphorylated U1-70K. This set of protocols promises to be a valuable resource for scientists exploring the intricate web of U1-70K-related mechanisms in the context of RNA splicing and its implications in neurodegenerative disorders and other disorders and biological processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of full-length U1-70K from E. coli Support Protocol 1: Making chemically competent BL21 Star pRARE/pBB535 cells Basic Protocol 2: Phosphorylation of full-length U1-70K using SRPK1 Support Protocol 2: Purification of SRPK1 Basic Protocol 3: Expression and purification of U1-70K BAD1 from E. coli Basic Protocol 4: Phosphorylation of U1-70K BAD1 using SRPK1 Basic Protocol 5: Expression and purification of U1-70K BAD2 from E. coli.

Purification of Adeno-Associated Viral Vector Serotype 9 Using Ceramic Hydroxyapatite Chromatography and its Analysis.

Adeno-associated virus (AAV) vectors can efficiently transduce exogenous genes into various tissues in vivo. Owing to their convenience, high efficiency, long-term stable gene expression, and minimal side effects, AAV vectors have become one of the gold standards for investigating gene functions in vivo, especially in non-clinical studies. However, challenges persist in efficiently preparing a substantial quantity of high-quality AAV vectors. Commercial AAV vectors are typically associated with high costs. Further, in-laboratory production is hindered by the lack of specific laboratory equipment, such as ultracentrifuges. Therefore, a simple, quick, and scalable preparation method for AAV vectors is needed for proof-of-concept experiments. Herein, we present an optimized method for producing and purifying high-quality AAV serotype 9 (AAV9) vectors using standard laboratory equipment and chromatography. Using ceramic hydroxyapatite as a mixed-mode chromatography medium can markedly increase the quality of purified AAV vectors. Basic Protocols and optional methods for evaluating purified AAV vectors are also described. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of AAV9 vectors in 293EB cells Basic Protocol 2: Concentration and buffer exchange of AAV9 vectors from 293EB cell culture supernatants using tangential flow filtration Basic Protocol 3: Purification of AAV9 vectors from TFF samples using ceramic hydroxyapatite chromatography Basic Protocol 4: Analysis of the purified AAV9 vectors.

GlycoID Proximity Labeling to Identify O-GlcNAcylated Protein Interactomes in Live Cells.

Cells continuously remodel their intracellular proteins with the monosaccharide O-linked N-acetylglucosamine (O-GlcNAc) to regulate metabolism, signaling, and stress. This protocol describes the use of GlycoID tools to capture O-GlcNAc dynamics in live cells. GlycoID constructs contain an O-GlcNAc binding domain linked to a proximity labeling domain and a subcellular localization sequence. When expressed in mammalian cells, GlycoID tracks changes in O-GlcNAc-modified proteins and their interactomes in response to chemical induction with biotin over time. Pairing the subcellular localization of GlycoID with the chemical induction of activity enables spatiotemporal studies of O-GlcNAc biology during cellular events such as insulin signaling. However, optimizing intracellular labeling experiments requires attention to several variables. Here, we describe two protocols to adapt GlycoID methods to a cell line and biological process of interest. Next, we describe how to conduct a semiquantitative proteomic analysis of O-GlcNAcylated proteins and their interactomes using insulin versus glucagon signaling as a sample application. This articles aims to establish baseline GlycoID protocols for new users and set the stage for widespread use over diverse cellular applications for the functional study of O-GlcNAc glycobiology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Expression of targeted GlycoID constructs to verify subcellular location and labeling activity in mammalian cells Basic Protocol 2: GlycoID labeling in live HeLa cells for O-GlcNAc proteomic comparisons.

Turning Trash Into Treasure: A Simple Protocol for Human Mesenchymal Stromal Cell Isolation From Used Bone Marrow Collection Kits.

The therapeutic potential of mesenchymal stromal cells (MSCs) has been extensively investigated in both preclinical and clinical settings. Recent years have witnessed the emergence of numerous isolation protocols and culture techniques, ranging from the selection of subpopulations to preserve stemness to preconditioning strategies aimed at enhancing therapeutic efficacy, tailored to the specific tissue source. In this protocol, we present a straightforward and cost-effective method for isolating human MSCs (hMSCs) from discarded bone marrow collection kits (comprising bag and filter systems) originally intended for removing impurities and unwanted cellular debris from the collected bone marrow aspirate, ensuring the purity of the stem cell population during stem cell transplantation. Utilizing basic laboratory equipment, we demonstrate the isolation of hMSCs, highlighting the expression of specific surface antigens, and multilineage differentiation into adipogenic, osteogenic, and chondrogenic lineages in vitro. This sustainable and resource-efficient approach not only contributes to reducing medical waste but also holds promise for advancing regenerative medicine applications. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of human mesenchymal stromal cells from bone marrow collection kits Basic Protocol 2: Culture of human mesenchymal stromal cells Basic Protocol 3: Characterization of human mesenchymal stromal cells with flow cytometry analysis Basic Protocol 4: Characterization of human mesenchymal stromal cells with multilineage differentiation under in vitro conditions.

Developing a Novel Enzalutamide-Resistant Prostate Cancer Model via AR F877L Mutation in LNCaP Cells.

Prostate cancer is a leading diagnosis and major cause of cancer-related deaths in men worldwide. As a typical hormone-responsive disease, prostate cancer is commonly managed with androgen deprivation therapy (ADT) to curb its progression and potential metastasis. Unfortunately, progression to castration-resistant prostate cancer (CRPC), a notably more aggressive phase of the disease, occurs within a timeframe of 2-3 years following ADT. Enzalutamide, a recognized androgen receptor (AR) antagonist, has been employed as a standard of care for men with metastatic castration-resistant prostate cancer (mCRPC) since it was first approved in 2012, due to its ability to prolong survival. However, scientific evidence suggests that sustained treatment with AR antagonists may induce acquired AR mutations or splice variants, such as AR F877L, T878A, and H875Y, leading to drug resistance and thereby diminishing the therapeutic efficacy of these agents. Thus, the establishment of prostate cancer models incorporating these particular mutations is essential for developing new therapeutic strategies to overcome such resistance and evaluate the efficacy of next-generation AR-targeting drugs. We have developed a CRISPR (clustered regularly interspaced short palindromic repeats)-based knock-in technology to introduce an additional F877L mutation in AR into the human prostate cell line LNCaP. This article provides comprehensive descriptions of the methodologies for cellular gene editing and establishment of an in vivo model. Using these methods, we successfully identified an enzalutamide-resistant phenotype in both in vitro and in vivo models. We also assessed the efficacy of target protein degraders (TPDs), such as ARV-110 and ARV-667, in both models, and the corresponding validation data are also included here. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Generation of AR F877L-mutated LNCaP cell line using CRISPR technology Basic Protocol 2: Validation of drug resistance in AR F877L-mutated LNCaP cell line using the 2D CTG assay Support Protocol: Testing of sgRNA efficiency in HEK 293 cells Basic Protocol 3: Validation of drug resistance in AR F877L-mutated LNCaP cell line in vivo.

Prediction of Acute Hepatotoxicity With Human Pluripotent Stem Cell-Derived Hepatic Organoids.

Recent development of hepatic organoids (HOs) derived from human pluripotent stem cells (hPSCs) provides an alternative in vitro model that can mimic the human liver detoxification pathway for drug safety assessment. By recapitulating the high level of maturity and drug-metabolizing capacity of the liver in a three-dimensional organoid culture, HOs may allow researchers to assess drug toxicity and metabolism more accurately than animal models or hepatocellular carcinoma cells. Although this promising potential has contributed to the development of various protocols, only a few protocols are available to generate functional HOs with guaranteed CYP450 enzymatic activity, the key feature driving toxic responses during drug metabolism. Based on previously published protocols, we describe an optimized culture method that can substantially increase the expression and activity of CYP450s, in particular CYP3A4, CYP2C9, and CYP2C19, in HOs. To generate mass-produced and highly reproducible HOs required as models for toxicity evaluation, we first generated hepatic endodermal organoids (HEOs) from hPSCs capable of in vitro proliferation and cryopreservation. The stepwise protocol includes generating HEOs as well as efficient methods to enhance CYP450 expression and activity in terminally differentiated HOs. Furthermore, we present a simple protocol for the assessment of HO cytotoxicity, one of the hallmarks of drug-induced acute hepatotoxicity. The protocols are relatively straightforward and can be successfully used by laboratories with basic experience in culturing hPSCs. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of hepatic endodermal organoids from human pluripotent stem cells Basic Protocol 2: Expansion and cryopreservation of hepatic endodermal organoids Basic Protocol 3: Differentiation of hepatic organoids from hepatic endodermal organoids Basic Protocol 4: Evaluation of hepatotoxicity using hepatic organoids Support Protocol: Human pluripotent stem cell culture.

Gene Delivery and Analysis of Optogenetic Induction of Lytic Cell Death.

Necroptosis is a form of inflammatory lytic cell death involving active cytokine production and plasma membrane rupture. Progression of necroptosis is tightly regulated in time and space, and its signaling outcomes can shape the local inflammatory environment of cells and tissues. Pharmacological induction of necroptosis is well established, but the diffusive nature of chemical death inducers makes it challenging to study cell-cell communication precisely during necroptosis. Receptor-interacting protein kinase 3, or RIPK3, is a crucial signaling component of necroptosis, acting as a crucial signaling node for both canonical and non-canonical necroptosis. RIPK3 oligomerization is crucial to the formation of the necrosome, which regulates plasma membrane rupture and cytokine production. Commonly used necroptosis inducers can activate multiple downstream signaling pathways, confounding the signaling outcomes of RIPK3-mediated necroptosis. Opsin-free optogenetic techniques may provide an alternative strategy to address this issue. Optogenetics uses light-sensitive protein-protein interaction to modulate cell signaling. Compared to chemical-based approaches, optogenetic strategies allow for spatiotemporal modulation of signal transduction in live cells and animals. We developed an optogenetic system that allows for ligand-free optical control of RIPK3 oligomerization and necroptosis. This article describes the sample preparation, experimental setup, and optimization required to achieve robust optogenetic induction of RIPK3-mediated necroptosis in colorectal HT-29 cells. We expect that this optogenetic system could provide valuable insights into the dynamic nature of lytic cell death. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of lentivirus encoding the optogenetic RIPK3 system Support Protocol: Quantification of the titer of lentivirus Basic Protocol 2: Culturing, chemical transfection, and lentivirus transduction of HT-29 cells Basic Protocol 3: Optimization of optogenetic stimulation conditions Basic Protocol 4: Time-stamped live-cell imaging of HT-29 lytic cell death Basic Protocol 5: Quantification of HT-29 lytic cell death.

Use of Lentivirus-Based Method for Establishing TK6 Human Cell Lines Expressing Cytochrome P450 and its Applications in Genotoxicity Testing.

The human lymphoblastoid cell line TK6 stands out as the most widely employed human cell line in genotoxicity testing, as recommended by various testing guidelines for in vitro assessments. Nevertheless, like many testing cell lines, TK6 lacks functional phase I drug-metabolizing enzymes crucial for chemical genotoxicity evaluations. This protocol introduces a lentivirus-based methodology for establishing a panel of TK6-derived cell lines, each expressing one of 14 cytochrome P450s (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7). The utilization of a lentiviral expression system ensures stable transduction, offering notable advantages such as sustained transgene expression, high transduction efficiency, positive selection feasibility, and user-friendly application. Additionally, we present a detailed procedure for validating the enhanced expression of each CYP in the established cell lines through real-time PCR, western blotting, and mass spectrometry analysis. Lastly, we exemplify the application of these CYP-expressing TK6 cell lines in genotoxicity testing, employing a flow-cytometry-based in vitro micronucleus test. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Lentivirus production and transduction for TK6 cells Support Protocol: Selecting a single clone of CYP-expressing TK6 cells Basic Protocol 2: Validation of CYP expression in TK6 cell lines Basic Protocol 3: Application of transduced cell lines in flow-cytometry-based micronucleus assay.

Isolation and Culturing of Primary Murine Chondroprogenitor Cells: A Mammalian Model of Chondrogenesis.

Embryonic limb bud-derived micromass cultures are valuable tools for investigating cartilage development, tissue engineering, and therapeutic strategies for cartilage-related disorders. This collection of fine-tuned protocols used in our laboratories outlines step-by-step procedures for the isolation, expansion, and differentiation of primary mouse limb bud cells into chondrogenic micromass cultures. Key aspects covered in these protocols include synchronized fertilization of mice (Basic Protocol 1), tissue dissection, cell isolation, micromass formation, and culture optimization parameters, such as cell density and medium composition (Basic Protocol 2). We describe techniques for characterizing the chondrogenic differentiation process by histological analysis (Basic Protocol 3). The protocols also address common challenges encountered during the process and provide troubleshooting strategies. This fine-tuned comprehensive protocol serves as a valuable resource for scientists working in the fields of developmental biology, cartilage tissue engineering, and regenerative medicine, offering an updated methodology for the study of efficient chondrogenic differentiation and cartilage tissue regeneration. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synchronized fertilization of mice Basic Protocol 2: Micromass culture of murine embryonic limb bud-derived cells Basic Protocol 3: Qualitative assessment of cartilage matrix production using Alcian blue staining.

Optimized Recombinant Expression and Purification of the SARS-CoV-2 Polymerase Complex.

An optimized protocol has been developed to express and purify the core RNA-dependent RNA polymerase (RdRP) complex from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The expression and purification of active core SARS-CoV-2 RdRp complex is challenging due to the complex multidomain fold of nsp12, and the assembly of the multimeric complex involving nsp7, nsp8, and nsp12. Our approach adapts a previously published method to express the core SARS-CoV-2 RdRP complex in Escherichia coli and combines it with a procedure to express the nsp12 fusion with maltose-binding protein in insect cells to promote the efficient assembly and purification of an enzymatically active core polymerase complex. The resulting method provides a reliable platform to produce milligram amounts of the polymerase complex with the expected 1:2:1 stoichiometry for nsp7, nsp8, and nsp12, respectively, following the removal of all affinity tags. This approach addresses some of the limitations of previously reported methods to provide a reliable source of the active polymerase complex for structure, function, and inhibition studies of the SARS-CoV-2 RdRP complex using recombinant plasmid constructs that have been deposited in the widely accessible Addgene repository. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and production of SARS-CoV-2 nsp7, nsp8, and nsp12 in E. coli cells Support Protocol: Establishment and maintenance of insect cell cultures Basic Protocol 2: Generation of recombinant baculovirus in Sf9 cells and production of nsp12 fusion protein in T. ni cells Basic Protocol 3: Purification of the SARS-CoV-2 core polymerase complex.

Downregulation of Hypoxia Inducible Factor-1α in Primary Human Natural Killer Cells Using Small Interfering RNA Delivery with ExPERT ATx by MaxCyte.

Natural killer (NK) cells are innate cytokine-producing and cytolytic effector lymphocytes. Their function is responsive to environmental factors, e.g., hypoxia, a frequent feature of inflamed tissues. Such responses require that the NK cells up-regulate HIF-1α (hypoxia inducible factor-1α), the major mediator of cellular responses to hypoxia that affects cell survival as well as immune responses. Thus, a major approach to the study of NK cell effector function under hypoxic conditions involves the ability to regulate HIF-1α levels in primary human NK cells. One difficulty with this approach, however, is that NK cells are difficult-to-transfect cells and common transfection methods, including electroporation or lipofection, suffer from variable transfection efficiency and cell viability. Moreover, the detection of HIF-1α is technically challenging because of the rapid degradation of the protein under normoxic conditions. Here, using the commercially available ExPERT ATx by MaxCyte, we report a workflow for the reliable delivery of small interfering RNA (siRNA) for targeting HIF-1α expression in primary human NK cells. We further provide a protocol for the detection of HIF-1α by immunoblot analysis demonstrating its efficient downregulation by siRNA. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of natural killer cells from human peripheral blood mononuclear cells Basic Protocol 2: Delivery of non-coding small interfering RNA and HIF-1α targeting siRNA into natural killer cells using ExPERT ATx Basic Protocol 3: Assessing the downregulation of HIF-1α protein using immunoblot analysis Support Protocol 1: Exemplary assessment of transfection efficiency using fluorescently labeled non-targeting siRNA Support Protocol 2: Exemplary assessment of NK cell viability 20 hr post-transfection Support Protocol 3: Exemplary assessment of HIF-1α knockdown using immunoblot analysis.

Three-Dimensional Cell Culture of Epididymal Basal Cells and Organoids: A Novel Tool for Toxicology.

Spermatozoa are formed in the testis but must transit through the epididymis to acquire motility and the ability to fertilize. The epididymis is a single convoluted tubule comprising several anatomically and physiologically distinct regions. The pseudostratified epithelium consists of multiple cell types, including principal cells, clear cells, narrow cells, and apical cells, that line the lumen of the epididymis. Basal cells are present at the base of the epithelium, and halo cells, which includes macrophages/monocytes, mononuclear phagocytes, and T lymphocytes, are also present in the epithelium. Several aspects of this complex spermatozoan maturation process are well established, but a great deal remains poorly understood. Given that dysfunction of the epididymis has been associated with male infertility, in vitro tools to study epididymal function and epididymal sperm maturation are required. Our lab and others have previously developed human, rat, and mouse epithelial principal cell lines, which have been used to address certain questions, such as about the regulation of junctional proteins in the epididymis, as well as the toxicity of nonylphenols. Given that the epididymal epithelium comprises multiple cell types, however, a 3D in vitro model provides a more comprehensive and realistic tool that can be used to study and elucidate the multiple aspects of epididymal function. The purpose of this article is to provide detailed information regarding the preparation, maintenance, passaging, and immunofluorescent staining of rat epididymal organoids derived from adult basal cells, which we have demonstrated to be a type of adult stem cell in the rat epididymis. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of epididymal cells Basic Protocol 2: Magnetic activated cell sorting and isolation of basal cells Basic Protocol 3: Preparation and culture of epididymal basal cell organoids Basic Protocol 4: Passage of epididymal basal cell organoids Basic Protocol 5: Freezing and thawing of epididymal basal cell organoids Basic Protocol 6: Immunofluorescent staining of epididymal basal cell organoids.

Current Strategies of Modeling Human Trophoblast Using Human Pluripotent Stem Cells in vitro.

We previously established a trophoblast differentiation protocol from primed human pluripotent stem cells (PSC). To induce this lineage, we use a combination of Bone Morphogenetic Protein-4 (BMP4) and the WNT inhibitor IWP2. This protocol has enabled us to obtain a pure population of trophectoderm (TE)-like cells that could subsequently be terminally differentiated into syncytiotrophoblasts (STB) and extravillous trophoblasts (EVT). However, the resulting TE-like cells could only be terminally differentiated to a variable mixture of STB and EVT, with a bias toward the STB lineage. Recently, methods have been developed for derivation and culture of self-renewing human trophoblast stem cells (TSC) from human embryos and early gestation placental tissues. These primary TSCs were further able to differentiate into either STB or EVT with high efficiency using the lineage specific differentiation protocols. Based partly on these protocols, we have developed methods for establishing self-renewing TSC-like cells from PSC, and for efficient lineage-specific terminal differentiation. Here, we describe in detail the protocols to derive and maintain PSC-TSC, from both embryonic stem cells (ESC) and patient-derived induced pluripotent stem cells (iPSC), and their subsequent terminal differentiation to STB and EVT. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Trophoblast Differentiation into TE-like Cells Basic Protocol 2: Conversion of PSC-Derived TE-like Cells to TSC Basic Protocol 3: Passaging PSC-Derived TSC in iCTB Complete Medium Basic Protocol 4: STB Differentiation from PSC-derived TSC Basic Protocol 5: EVT Differentiation from PSC-derived TSC Support Protocol 1: Geltrex-coated tissue culture plate preparation Support Protocol 2: Collagen IV-coated tissue culture plate preparation Support Protocol 3: Fibronectin-coated tissue culture plate preparation.

Measurement of Trogocytosis: Quantitative Analyses Validated with Rigorous Controls.

Trogocytosis is a process in which receptors on acceptor cells remove and internalize cognate ligands from donor cells. Trogocytosis has a profound and negative impact on mAb-based cancer immunotherapy, as seen in the treatment of chronic lymphocytic leukemia (CLL) with CD20 mAbs, such as rituximab (RTX) and ofatumumab (OFA). Our clinical observations of RTX/OFA-mediated loss of the CD20 target from circulating CLL cells have been replicated in our in vitro studies. Here we describe flow cytometry and fluorescence microscopy experiments, which demonstrate that acceptor cells, such as monocytes/macrophages that express FcγR, remove and internalize both antigen and donor cell-bound cognate IgG mAbs for several different mAb-donor cell pairs. Fluorescent mAbs and portions of the plasma cell membrane are transferred from donor cells to acceptor cells, which include the THP-1 monocytic cell line as well as freshly isolated monocytes. We describe rigorous controls to validate the reactions and eliminate dissociation or internalization as alternative mechanisms. Trogocytosis is likely to contribute to neutropenia, thrombocytopenia, and liver damage associated with use of antibody-drug conjugates. The methods we have described should allow for examination of strategies focused on blocking trogocytosis and its adverse effects. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Trogocytosis of mAb-opsonized donor cells mediated by adherent THP-1 cells Alternate Protocol: Application of fluorescence microscopy to examine THP-1 cell-mediated trogocytosis Support Protocol 1: Alexa labeling of mAbs and determination of F/P ratios Support Protocol 2: Standard washing procedure Support Protocol 3: Labeling and opsonization of cells Basic Protocol 2: Trogocytosis mediated by human monocytes as acceptor cells Support Protocol 4: Isolation of human monocytes Basic Protocol 3: Trogocytosis mediated by THP-1 cells in solution Support Protocol 5: Retinoic acid treatment of THP-1 cells Support Protocol 6: Culturing of SCC-25, BT-474, MOLT-4 and THP-1 cell lines.