Current protocols最新文献

Coronaviridae spike glycoproteins mediate viral entry and fusion to host cells through binding to host receptors (i.e., ACE2, DPP4) and are key components in determining viral host range, making them targets for antiviral research. Here, we describe the expression, purification, and characterization of recombinant spike proteins to aid in protein characterization and analysis. These protocols were used for the production of spike glycoproteins from civet, pangolin, and bat coronaviruses, as well as high-resolution cryo-electron microscopy (cryo-EM) structural analysis of bat and civet host coronavirus spike glycoproteins (Hills et al., 2024). © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Expression and purification of SARS-CoV spike protein from ExpiCHO cells

Basic Protocol 2: Preparation of SARS-CoV spike protein for visualization by negative-stain transmission electron microscopy and cryo-electron microscopy

Tuberculosis (TB) remains one of the leading infectious causes of death worldwide. Persistent bacterial populations in specific microenvironments within the host hamper efficient TB chemotherapy. Caseum in the necrotic core of closed granulomas and cavities of pulmonary TB patients can harbor high burdens of drug-tolerant Mycobacterium tuberculosis (MTB) bacilli, making them particularly difficult to sterilize. Here, we describe protocols for the generation of a surrogate matrix using lipid-rich macrophages to mimic the unique composition of caseum in vivo. Importantly, this caseum surrogate induces metabolic and physiological changes within MTB that reproduce the nonreplicating drug-tolerant phenotype of the pathogen in the native caseous environment, making it advantageous over alternative in vitro models of nonreplicating persistent (NRP) MTB. The protocols include culture of THP-1 monocytes, stimulation of lipid droplet accumulation, lysis and denaturation of the foamy macrophages, inoculation and preadaptation of MTB bacilli in the caseum surrogate, and evaluation of drug bactericidal activity against the NRP population. This novel in vitro model is being used to screen for potent bactericidal antimicrobial agents and to identify vulnerable drug targets, among a variety of other applications, thereby reducing our reliance on in vivo models. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Caseum surrogate preparation from γ-irradiated M. tuberculosis–induced foamy THP-1 monocyte–derived macrophages (THPMs)

Alternate Protocol 1: Caseum surrogate preparation from stearic acid–induced THPMs

Basic Protocol 2: Generation of nonreplicating persistent M. tuberculosis and drug susceptibility testing

Alternate Protocol 2: Higher-throughput drug susceptibility screening using caseum surrogate

Induced pluripotent stem cells (iPSCs) have revolutionized the fields of regenerative medicine, disease modeling, and drug discovery. However, the usage of iPSCs for various applications has been hampered by the observed line-to-line variability in their differentiation capacity. Therefore, it is important to verify the pluripotent status of iPSCs. A very effective way to define the pluripotent state of iPSCs is by evaluating the expression of established undifferentiated stem cell markers. A bona fide iPSC must have high, homogeneous expression of these markers. Here, we present a cost-effective platform that can be readily utilized by researchers to define the pluripotency status of iPSCs by measuring the expression of surface and intracellular markers by flow cytometry. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: iPSC culture and collection for flow cytometry analysis

Basic Protocol 2: Staining of iPSCs for extracellular and intracellular undifferentiated stem cell markers

Basic Protocol 3: Flow cytometry acquisition

Basic Protocol 4: Flow cytometry data analysis

Paclitaxel, one of the most commonly used anticancer agents, is employed in the treatment of a range of malignant tumors. However, resistance is one of the major barriers to successful therapy. Despite its clinical relevance, the molecular mechanisms underlying paclitaxel resistance remain poorly understood. In this protocol, we describe the methods for establishing paclitaxel-resistant tumor models both in vitro and in vivo, and how we investigated the underlying mechanisms of resistance. Additionally, we evaluated the potential of combination therapies to overcome paclitaxel resistance in these models. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Generation of paclitaxel-resistant breast cancer model in vivo.

Basic Protocol 2: Generation of paclitaxel-resistant gastric cancer model in vitro.

Basic Protocol 3: Validation of drug resistance in vivo.

Basic Protocol 4: In vitro and in vivo evaluation of combination therapy in paclitaxel-resistant models.

Multiplexed error-robust fluorescence in situ hybridization (MERFISH) is a massively multiplexed single RNA–molecule imaging technique capable of spatially resolved single-cell transcriptomic profiling of thousands of genes in millions of cells within intact tissue slices. Initially introduced for brain tissues, MERFISH has since been extended to other tissues, where rapid RNA degradation during the preparation process can pose challenges. This protocol outlines the application of MERFISH in one such challenging tissue, the mammalian gastrointestinal tract. We describe two complementary protocols leveraging either fresh frozen or fixed frozen approaches and describe methods for combining RNA imaging with immunofluorescence. While these protocols were designed and validated in gut tissues, we anticipate that they will be useful resources for the application to other challenging tissue types. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Fixed-frozen sample preparation

Basic Protocol 2: Fresh-frozen sample preparation

Basic Protocol 3: Encoding probe construction

Basic Protocol 4: MERFISH imaging

Basic Protocol 5: Image decoding

Support Protocol 1: Coverslip silanization

Support Protocol 2: Poly-d-lysine (PDL) coating of the coverslips

Support Protocol 3: Hybridization buffer preparation

Support Protocol 4: Trolox quinone stock preparation

Support Protocol 5: TCEP stock preparation

Alternate Protocol 1: MERFISH-compatible immunofluorescent boundary stains in fresh frozen tissue

Alternate Protocol 2: Immunofluorescent boundary stains with methacrylate-NHS-anchored antibodies for PFA-fixed samples

Alternate Protocol 3: Guanidine-HCl tissue clearing

This article highlights experimental procedures and troubleshooting tips for the utilization of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) methods for detecting and visualizing lipid alterations in the mouse brain tissue in response to efavirenz (EFV) treatment. To investigate drug-induced adverse effects, it is becoming increasingly important to understand the spatial alterations of lipid molecules in the target organs. EFV is a non-nucleoside reverse transcriptase inhibitor commonly used for HIV treatment in combination with other antiretrovirals. Importantly, EFV is a drug that is included in the World Health Organization's list of essential medications. However, EFV is known to be associated with neurotoxicity. To date, the mechanisms underlying EFV-induced neurotoxicity have not been fully elucidated. Therefore, it is important to gain understanding of the effect of EFV on the brain. It is known that the brain is composed of different neuroanatomical regions that are abundant in lipids. Described here is the use of a chemical imaging strategy, MALDI MSI, to detect, identify, and visualize the spatial localization of several lipid species across the brain tissue sections along with their alterations in response to EFV treatment. The set of protocols consists of three major parts: lipid detection, identification, and tissue imaging. Lipid detection includes testing different chemical matrices and how they facilitate the detection of analytes, which is then followed by identification. Collision-induced dissociation is employed to verify the identity of the lipid molecules. Lastly, tissue imaging experiments are performed to generate the spatial localization profiles of the lipids. The protocols described in this article can be employed to spatially visualize alterations in the lipid molecules in response to drug treatment. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: MALDI mass spectrometry (MALDI MS) profiling experiments for detection of lipids

Basic Protocol 2: MALDI MS imaging of lipid molecules in mouse brain tissues

Basic Protocol 3: MALDI MS data processing and analysis

Mice carrying patient-associated base changes are powerful tools to define the causality of single-nucleotide variants to disease states. Epitope tags enable immuno-based studies of genes for which no antibodies are available. These alleles enable detailed and precise developmental, mechanistic, and translational research. The first step in generating these alleles is to identify within the target sequence—the orthologous sequence for base changes or the N or C terminus for epitope tags—appropriate Cas9 protospacer sequences. Subsequent steps include design and acquisition of a single-stranded oligonucleotide repair template, synthesis of a single guide RNA (sgRNA), collection of zygotes, and microinjection or electroporation of zygotes with Cas9 mRNA or protein, sgRNA, and repair template followed by screening born mice for the presence of the desired sequence change. Quality control of mouse lines includes screening for random or multicopy insertions of the repair template and, depending on sgRNA sequence, off-target sequence variation introduced by Cas9. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Single guide RNA design and synthesis

Alternate Protocol 1: Single guide RNA synthesis by primer extension and in vitro transcription

Basic Protocol 2: Design of oligonucleotide repair template

Basic Protocol 3: Preparation of RNA mixture for microinjection

Support Protocol 1: Preparation of microinjection buffer

Alternate Protocol 2: Preparation of RNP complexes for electroporation

Basic Protocol 4: Collection and preparation of mouse zygotes for microinjection or electroporation

Basic Protocol 5: Electroporation of Cas9 RNP into zygotes using cuvettes

Alternate Protocol 3: Electroporation of Cas9 RNP into zygotes using electrode slides

Basic Protocol 6: Screening and quality control of derived mice

Support Protocol 2: Deconvoluting multiple sequence chromatograms with DECODR

Glioblastoma (GBM) carries a dismal prognosis, with a median survival of less than 15 months. Temozolomide (TMZ), the standard frontline chemotherapeutic for GBM, is an alkylating agent that generates DNA O⁶-methylguanine (O⁶MeG) lesions. Without O⁶MeG-methyltransferase (MGMT), this lesion triggers the mismatch repair (MMR) pathway and leads to cytotoxicity via futile cycling. TMZ resistance frequently arises via the somatic acquisition of MMR deficiency (MMRd). Moreover, DNA-damaging agents have been shown capable of increasing tumor immunogenicity and improving response to immune checkpoint blockade (ICB), which has had limited success in glioma. The study of how alkylating chemotherapy such as TMZ impacts antitumor immunity in glioma has been hindered by a lack of immunocompetent models that incorporate relevant DNA repair genotypes. Here, we used CRISPR/Cas9 to generate models isogenic for knockout (KO) of Mlh1 in the syngeneic SB28 murine glioma cell line. MMR KO models readily formed intracranial tumors and exhibited in vitro and in vivo resistance to TMZ. In contrast, MMR KO cells maintained sensitivity to KL-50, a newly developed alkylating compound that exerts MGMT-dependent, MMR-independent cytotoxicity. Lastly, MMR KO tumors remained resistant to ICB, mirroring the lack of response seen in patients with somatic MMRd GBM. The development of syngeneic, immunologically cold glioma models with somatic loss of MMR will facilitate future studies on the immunomodulatory effects of alkylating agents in relevant DNA repair contexts, which will be vital for optimizing combinations with ICB. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Validation of mismatch repair knockouts and in vitro sensitivity to alkylating agents

Basic Protocol 2: Stereotaxic injection of isogenic SB28 cells in female C57BL/6J mice and in vivo treatment

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

Gut mucosae are composed of stratified layers of microbes, a selectively permeable mucus, an epithelial lining, and connective tissue homing immune cells. Studying cellular and chemical interactions between the gut mucosal components has been limited without a good model system. We have engineered a three-dimensional (3D) multi-cellular co-culture system we coined “3D Flipwell system” using cell culture inserts stacked against each other. This system allows an assessment of the impact of a gut mucosal environmental change on interactions between gut bacteria, epithelia, and immune cells. As such, this system can be utilized in examining the effects of exogenous stimuli, such as dietary nutrients, bacterial infection, and drugs, on the gut mucosa that could predetermine how these stimuli might influence the rest of body. Here, we describe the methods of construction and application of the new 3D Flipwell system we utilized previously in assessing the crosstalk between the gut mucosa and macrophage polarization. We demonstrate the physiological responses of different components of the co-cultures to Sepiapterin (SEP), the precursor of the nitric oxide synthase cofactor tetrahydrobiopterin (BH₄). We reported previously that SEP induces a pro-immunogenic shift of macrophages having acquired an immune suppressive phenotype. We also showed that SEP induces a defense mechanism of commensal gut bacteria. The protocol describing the assembly and use of the 3D Flipwell co-culture system herein would grant its utility in evaluating the concurrent effects of pharmacologic and microbiologic stimuli on gut mucosal components. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: 3D Flipwell construction, assembly, and collagen coating

Basic Protocol 2: Flipwell cell seeding and cell culture

Basic Protocol 3: Addition of bacterial culture to the Flipwell system

Basic Protocol 4: Flipwell disassembly for scanning electron microscopy (SEM) studies

Basic Protocol 5: Immunofluorescence antibody staining for confocal microscopy