Current protocols最新文献

Renal ischemia-reperfusion injury (IRI), one of the most important causes of acute kidney injury, is a significant health problem that can result in acute kidney failure after surgical procedures in which blood flow to the kidney is temporarily interrupted. Although numerous studies have been conducted to prevent renal IRI damage, this problem remains unresolved due to its complex nature. During IRI, both ischemia and reperfusion contribute to damage, with oxidative stress, inflammation, and the activation of apoptotic mechanisms playing significant roles in exacerbating the damage. This article provides comprehensive protocols for inducing renal IRI in rats, including both bilateral and unilateral models. These protocols can be used in studies designed to understand the cellular and molecular mechanisms of renal IRI and exploring potential therapeutic strategies. When the described procedure is carefully applied in experimental IRI models, the mortality rate in animals will be lower. Furthermore, we outline opportunities for investigating oxidative stress, inflammatory and apoptotic pathways, and highlight how these analyses may contribute to preclinical therapeutic studies. We also provide recommendations on which analyses should be performed in such studies. Specifically, information is provided on which kidney damage biomarkers should be examined in samples obtained from animals. Additionally, we explain which investigations can be performed on apoptotic and inflammatory pathways involved in kidney damage. In summary, this study integrates bilateral and unilateral approaches and provides a detailed methodological framework for mechanistic and therapeutic research on renal ischemia-reperfusion injury. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Bilateral kidney ischemia-reperfusion in rats

Alternate Protocol: Unilateral kidney ischemia-reperfusion in rats

Basic Protocol 2: Analyses performed on kidney tissues and serum of rats after ischemia-reperfusion

Understanding a protein's interactome provides crucial insight into its function and its contribution to disease. Traditional methods such as co-immunoprecipitations often fail to capture interactions that are dependent on native cellular architecture such as those found on the cellular membrane. While enzyme-based proximity labeling utilizing peroxidases or biotin ligases can achieve in situ interactome mapping, these approaches are limited by labeling radii, cellular engineering, and amino acid labeling biases. Antibody-guided µMap photoproximity labeling addresses the constraints of these alternative platforms. Here, we apply µMap photoproximity labeling to study the interactome of HER2 by using antibody-guided proximity labeling to target the endogenous protein, ablating the need for cellular engineering, and leveraging the advantages of µMap's short 4-nm labeling radius. The protocols presented here describe the preparation of iridium-antibody conjugates and its application in studying protein interactomes through mass-spectrometry based analysis. While HER2 was used as a model in this article, this method is broadly applicable and can be used to study any cell surface protein with an appropriate commercially available antibody. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Preparation and validation of iridium-antibody conjugates

Basic Protocol 2: Proximity labeling and streptavidin enrichment for mass spectrometry

This article describes the isolation, culture, and integration of primary murine osteocytes on a microfluidic platform for in vitro mechanobiology studies. Osteocytes are mechanosensitive cells embedded within the bone matrix and regulate bone remodeling. The MLO-Y4 osteocyte-like cell line typically used for mechanobiology studies can provide a good physiological response to mechanical stimulation but has been shown to have missing or low levels of key osteocyte markers found in vivo, such as sclerostin. However, primary osteocytes are difficult to isolate and study in vitro due to their location within the bone matrix, and the scope of existing protocols for osteocyte extraction only includes the isolation of primary cells using collagenase. Here, optimized protocols for the isolation, culture, and real-time calcium imaging of primary osteocytes in response to oscillatory fluid flow are outlined. Moreover, we discuss how to incorporate and maintain primary osteocytes on a physiologically relevant microfluidic platform for in vitro bone metastasis studies. In calcium imaging and microfluidic experiments, primary osteocytes are compared to the MLO-Y4 cell line as a control or reference group. Primary osteocytes can be extracted in 6 to 8 hr and typically require 1 week to recover before use in experiments. Calcium imaging of primary osteocytes can be completed in 24 hr. The time required for microfluidic culture varies on the platform used; the microfluidic experiment described here takes 6 days to complete. We anticipate that the information from this protocol will aid other researchers with incorporating primary osteocytes to enhance the biological relevance of in vitro studies on bone mechanobiology and disease. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Isolation of primary murine osteocytes using Liberase TM

Basic Protocol 2: Primary osteocyte subculture and E11/podoplanin staining

Basic Protocol 3: Real-time calcium imaging of primary osteocytes

Basic Protocol 4: Integration of primary murine osteocytes onto a microfluidic platform for observing bone metastasis

We present here a new reagent enabling the supported synthesis of oligodeoxynucleotides (ODNs) and oligoribonucleotides (ORNs) containing a phosphate group at the 5′-terminal position after complete deprotection. This reagent, derived from a dihydroxyacetone core, contains a dimethoxytrityl (DMTr) group. The procedure for final deprotection is very similar to that routinely used in the synthesis of unmodified ODNs or ORNs. In particular, it preserves the advantages of the “trityl-on” method, namely facile purification by reverse-phase high-performance liquid chromatography (RP-HPLC), short treatment with an acetic acid solution in water, the possibility of on-resin monitoring by trityl cation assay, and the use of equipment commonly founded in chemical laboratories. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Phosphorylation of oligodeoxyribonucleotides (ODNs)

Alternate Protocol: Phosphorylation of oligoribonucleotides (ORNs)

Support Protocol: Preparation of phosphorylation reagent 1

High-precision genome editing tools, such as programmable nucleases, are poised to transform crop breeding and significantly impact fundamental plant research. Among these tools, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated 9) system is a programmable, RNA-guided nuclease that introduces targeted, site-specific double-stranded breaks in the target DNA loci. When these breaks are repaired, it often results in a frame-shift mutation via short insertion/deletion (indel), leading to gene knockout. Since its first successful use in plants, CRISPR/Cas9 has been widely adopted for targeting genes of agronomic and scientific importance in multiple crops, including rice, maize, wheat, and sorghum. These cereal crops ensure global food security, provide essential nutrition, and support economic stability. Additionally, such crops support biofuel production, livestock feed, and sustainable farming practices through crop rotation. This article outlines the strategies for implementing CRISPR/Cas9 genome editing in plants, including a step-by-step process of guide RNA target selection, oligonucleotide design, construct development, assembly, and analysis of genome edits. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: CRISPR/Cas9 guide RNA target selection

Support Protocol 1: Genomic DNA extraction in-house protocol

Basic Protocol 2: Construction of a binary plasmid vector

Support Protocol 2: Agrobacterium transformation with a binary vector construct and stability check

Support Protocol 3: Plant transformation

Basic Protocol 3: Genotyping of edited events

The cover image is based on the article CRISPR/Cas9-mediated gene knockout in cereal crops by Dibyajyoti Pramanik et al., https://doi.org/10.1002/cpz1.70210.

High quality input data is the key to successful interpretation of any scientific assay. In flow cytometry, fluorescently-conjugated antibodies allow us to simultaneously measure an incredible range of protein-based targets on single cells with a high degree of specificity. Limiting the quality of data generated, however, is the non-specific interaction that can occur between antibodies and off-target binders. Judicious use of blocking reagents can improve the specificity of the staining by reducing this non-specific binding to cells, improving the sensitivity of the assay to detect the authentic signal above assay noise. Additional beneficial effects include preventing interactions between dyes and even limiting the degradation of dyes, improving data quality. In this article, we provide a workflow for minimizing these unwanted effects, increasing specificity and sensitivity in highly multiplex flow cytometry. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Surface staining

Basic Protocol 2: Intracellular staining

Basic Protocol 3: Intracellular cytokine staining

Transplantation is the definitive treatment for patients with end-stage organ failure. Following allogeneic transplant, the recipient's immune system recognizes transplanted cells or organs as foreign. The immune system recognizes and targets the foreign tissue for damage through cell-mediated rejection (CMR) and/or antibody-mediated rejection (AMR). Immunosuppressive agents are utilized to protect the transplant from rejection and extend transplant function and survival. Despite advances in immunosuppressive agents, AMR remains a critical barrier to the success of transplantation. AMR occurs when B cells produce alloantibodies that bind the allograft causing antibody-dependent, complement-mediated or immune cell-mediated cytotoxic damage. Continued research on AMR is required to develop novel and effective therapeutic strategies. Murine AMR models have been utilized to investigate mechanisms mediating the production of posttransplant alloantibodies and the pathology of damage to the transplanted allograft. These models facilitating the investigation of cellular and molecular mechanisms of alloantibody production and allograft damage are critical to the development of novel therapeutic strategies to prevent and treat AMR. This article describes the methodologies used to study AMR in animal transplant models. These include protocols to detect and measure alloantibodies, allograft survival, AMR pathology, and effector immune cell responses following transplantation. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Alloserum transfer into immune-incompetent recipient mice to determine transplant organ susceptibility to AMR

Basic Protocol 2: Allogeneic transplantation into immune-deficient mice to study critical cellular and molecular pathways impacting AMR

Support Protocol 1: Quantification of posttransplant alloantibody titer

Support Protocol 2: Monitoring of transplant allograft survival

Support Protocol 3: Assessment of immunopathology and severity of AMR

Basic Protocol 3: Analysis of posttransplant immunologic responses

Since the birth of biochemistry, researchers have investigated the structure–function relationship of a wide variety of proteins. However, until recently, when X-ray free-electron lasers (XFELs) became available, it was not possible to visualize the motion of proteins from moment to moment with excellent temporal and spatial resolution. Here, we introduce practical methods to visualize protein motions at room temperature using serial femtosecond crystallography (SFX) using XFELs. With the development of this technology, it will be possible to visualize the entire reaction mechanism of many proteins in the future. We first outline a streamlined microcrystallization workflow for hen egg-white lysozyme, enabling rapid detector calibration and data-collection optimization. Next, we present a rotational seeding approach refined on copper-containing nitrite reductase that yields homogeneous microcrystals suitable for high-resolution SFX and readily adaptable to other challenging targets. Finally, we describe a time-resolved strategy combining microcrystals of fungal nitric-oxide reductase with photolabile caged substrates and synchronized UV triggering, capturing catalytic intermediates on the millisecond timescale. Together, these procedures enable investigators to progress from preparing samples to capturing dynamic structural snapshots. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Microcrystallization of lysozyme

Basic Protocol 2: Microcrystallization of copper-containing nitrite reductase

Basic Protocol 3: Time-resolved serial femtosecond crystallography

Acinetobacter baumannii is a high-risk pathogen associated with increased patient morbidity and mortality. Host-pathogen interactions amplify its virulence, in part by promoting biofilm formation—a crucial factor in antimicrobial resistance and persistence. Given the bacterium's strong propensity for acquiring resistance, antimicrobial susceptibility testing (AST) is essential for guiding effective therapeutic interventions. However, discrepancies have been observed between in vitro AST results and therapeutic outcomes, with some antimicrobials being deemed to show in vivo efficacy despite appearing ineffective in vitro. This discordance may stem from traditional AST protocols, which rely on bacteriological media such as Mueller Hinton broth (MHB) optimized for bacterial growth but not for mimicking the host environment. Moreover, conventional AST does not account for virulence traits such as biofilm formation, which further contribute to treatment failure. Incorporating physiologically relevant culture media, such as Roswell Park Memorial Institute (RPMI) 1640 medium, alongside assessment of biofilm formation may improve the predictive value of AST. This work outlines two complementary protocols for improving AST interpretation in A. baumannii infections. Basic Protocol 1 compares minimum inhibitory concentration (MIC) values generated using MHB and RPMI. Basic Protocol 2 evaluates biofilm formation in MHB, tryptic soy broth (TSB; control), and RPMI, with and without antimicrobial exposure. Together, these approaches aim to inform alternative AST strategies that better reflect in vivo conditions and optimize therapeutic decision-making. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Comparing A. baumannii minimum inhibitory concentration (MIC) results in bacteriological (MHB) versus physiological (RPMI) media

Basic Protocol 2: Comparing A. baumannii isolate(s) biofilm formation following assays completed in bacteriological culture media (MHB and control TSB) and physiological medium (RPMI)