Current protocols最新文献

Energy is required for life, and organisms obtain their energy from fuel sources to enable both anabolic and catabolic processes. Some of this energy is radiated as heat, which can be quantified as a measure of metabolic rate. In some cases, environmental toxicants can alter metabolic energy in undesirable ways, and characterization of new pharmaceuticals can determine the efficacy of desirable metabolic rate manipulation or identify off-target adverse effects. Current methods to directly measure heat production in laboratory mice are expensive, can be laborious, and make it challenging to monitor animals in ways that are multiplexed, robust, and non-invasive. We present a set of protocols for assembling and deploying a simple, low-cost thermal camera to monitor and record thermogenic activity, modified from prior work. Parts used to build this system currently cost approximately $150 USD and, when assembled, can record mouse temperatures as well as ambient cage temperatures up to twice per second for extended periods. By using multiplexed cameras in a diurnal mouse incubator system, the thermogenic capacity of several mice can be simultaneously recorded and graphed. Exogenous agents and genotypes that alter metabolism can be readily identified with this technology. In this set of protocols, we describe the assembly of the thermal video camera device, its use, and related data capture and analysis methods. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Assembling thermal camera for thermogenic stress test

Basic Protocol 2: In vivo measurement of mouse temperature under different ambient conditions

Site-directed mutagenesis is an essential tool in molecular biology, protein engineering, plasmid engineering and synthetic biology. While the QuickChange method has been one of the most employed methods for site-directed mutagenesis, it is hindered by low efficiency and frequent introduction of unwanted mutations at the primer sites, raising the urgent need for new, more efficient, and reliable methods. Here, we present an optimized site-directed mutagenesis protocol that leverages partially complementary primer pairs with 3’-overhangs to improve mutagenesis efficiency and reduce error rates. Our method significantly enhances success rates, achieving an average efficiency of ∼50% with some instances approaching the ideal threshold of 100%, while also minimizing the time required for mutant generation. Typically, only 3 colonies need to be analyzed per mutagenesis reaction, and a skillful trainee can engineer 1 to 2 dozen mutant plasmids within a week. In addition, with an in-house protocol for preparing highly competent bacterial cells, we have further increased the reliability and cost-effectiveness of the method. Notably, such competent cells have been kept in a liquid nitrogen tank for >12 years with minimal loss of competency. Thus, this refined method offers a robust, efficient, and scalable solution for high-precision gene modification in vitro, with broad applications in protein and plasmid engineering. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: In vitro site-directed mutagenesis using an optimized primer design strategy

Basic Protocol 2: Preparation of high-efficiency chemocompetent DH5α cells for transformation of mutagenized plasmid products

Basic Protocol 3: Transformation of chemocompetent DH5α cells and obtaining bacterial colonies with correctly mutagenized plasmid products

Alternate Protocol: Transformation if glycerol stocks are unavailable

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have become a powerful source for the in vitro modeling of cardiac diseases and various other essential applications, including cardiotoxicity screening and regenerative cell replacement therapies. Although many differentiation protocols have been developed to generate cardiomyocytes from human pluripotent stem cells, these protocols are costly and complex, requiring expensive and often unnecessary components (e.g., B27 medium supplement). In addition, the use of animal-derived growth factors limits their use for regenerative medicine purposes. To address these issues, herein, we have developed an efficient, cost-effective, and protein-free hPSC-CM protocol using only two components: DMEM/F12 basal medium and l-ascorbic acid 2-phosphate. By eliminating xenobiotic and complex components, the efficiency of directed differentiations is increased, the variability between cardiac differentiations is decreased, and the scalability of cell production is enhanced. Adaptation of this efficient, low-cost, and user-friendly cardiac differentiation protocol will enrich the utility and applicability of hPSC-CMs in drug discovery, cell therapies, tissue engineering, disease modeling, precision medicine, and cardiac regenerative medicine. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: hPSC cell culture

Basic Protocol 2: hPSC-CM differentiation

Basic Protocol 3: Characterization of hPSC-CMs by immunofluorescence (IF) imaging

Epimutation is defined as any heritable change in gene activity that does not affect the actual sequence of DNA. One of the most researched epimutations is promoter methylation of breast cancer susceptibility gene BRCA1. This epimutation may arise de novo in somatic tissues, such as breast or ovarian cancer tissue, but has also been shown to be widely distributed throughout tissues. BRCA1 methylation detectable in peripheral blood has been associated with the risk of both breast and ovarian cancer in patients without a germline pathogenic variant status of this gene. The strength of this association suggests that diagnostic screening for the BRCA1 epimutation should be considered with potential implications in cancer risk assessment. However, the detection of BRCA1 epimutation may be challenging, as it is generally detectable at a very low level in blood. Methylation-sensitive high-resolution melting (MS-HRM) was shown to provide a high sensitivity of methylation detection, and as it is a PCR-based method, epimutation screening based on MS-HRM is labor- and cost-effective. Here we describe the MS-HRM protocol for BRCA1 epimutation screening. © 2025 Wiley Periodicals LLC.

Basic Protocol: High sensitivity constitutional epimutations testing with MS-HRM

Porphyrins and porphyrin precursors are normally detected in small amounts in healthy individuals but are found in large quantities in the urine, feces, blood, plasma, bone marrow, and liver in patients with various types of porphyrias. These are intermediates, or are derived from intermediates, in the pathway for heme biosynthesis. Heme is synthesized in all body tissues but in the largest amounts in the bone marrow and liver. Accurately measuring these compounds is important for diagnosis and monitoring of porphyrias. In addition, measurement of enzyme activities and mutation analyses by DNA sequencing enables confirmation of a porphyria diagnosis and genetic counseling. Biochemical approaches described here include measurements of porphyrin precursors and porphyrins in the urine, feces, plasma, erythrocytes, and liver, and determination of specific enzyme activities in erythrocytes and other cells. © 2025 Wiley Periodicals LLC.

Hemostasis is a defense mechanism for preventing fluid loss in an injured organism with a circulatory system. In mammals, it begins at the site of the injury, with platelet adhesion to components of the subendothelial matrix activating a series of platelet signaling events that ultimately form a primary hemostatic plug. This process is followed by coagulation cascade activation, leading to fibrin formation and reinforcement of the plug. Thrombosis, an intravascular form of fibrin formation, is unpredictable and often associated with severe pain. Despite decades of research, many hemostatic and thrombotic factors remain unknown. To address this, we introduced zebrafish as a genetic model for studying thrombosis and pain. The piggyback knockdown method is used to knock down genes related to hemostasis and other biochemical or physiological pathways. Blood collection in zebrafish is important in characterizing hemostasis mutants such as hemophilia, in assaying blood coagulation, and in counting thrombocytes in diseases such as thrombocytopenia. FACS analysis is used to study thrombocyte development. Thrombocyte aggregation by flow cytometry and a plate tilt method is used in the functional evaluation of thrombocytes in hemostasis. Defects in coagulation can be studied by kinetic blood coagulation assays and bleeding assays. Laser thrombosis is a global assay that measures blood clotting, thrombocyte function, and endothelial function, including stasis. This technique is useful when screening for genes affecting hemostasis in a genome-wide manner. As pain is sensed by the PAR2 receptor, the trypsin aversion assay detects this pathway and can be used to identify genes related to pain pathways. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Gene silencing by piggyback knockdown

Basic Protocol 2: Blood collection from adult zebrafish

Basic Protocol 3: Quantification of thrombocytes by FACS analysis

Basic Protocol 4: Thrombocyte aggregation assay by flow cytometry

Alternate Protocol 1: Whole-blood aggregation assay by the plate tilt method

Basic Protocol 5: Kinetic blood coagulation assays

Support Protocol: Preparation of zebrafish thromboplastin

Basic Protocol 6: Laser thrombosis assay

Basic Protocol 7: Chemically induced gill bleeding assay

Alternate Protocol 2: Mechanically induced bleeding assay

Basic Protocol 8: Trypsin aversion assay in zebrafish larvae

Proximity ligation assays (PLAs) use specific antibodies to detect endogenous protein-protein interactions. PLAs are a highly useful biochemical technique that allow two proteins within proximity to be visualized with fluorescent probes amplified by PCR. While this technique has gained prominence, the use of a PLA in mouse skeletal muscle (SkM) is novel. In this article, we discuss how the PLA method can be used in SkM to study the protein-protein interactions within mitochondria-endoplasmic reticulum contact sites (MERCs). © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Proximity ligation assay for skeletal muscle tissue and myoblast for MERC proteins

OPA1 is a dynamin-related GTPase that modulates mitochondrial dynamics and cristae integrity. Humans carry eight different isoforms of OPA1 and mice carry five, all of which are expressed as short- or long-form isoforms. These isoforms contribute to OPA1's ability to control mitochondrial energetics and DNA maintenance. However, western blot isolation of all long and short isoforms of OPA1 can be difficult. To address this issue, we developed an optimized western blot protocol based on improving running time to isolate five different isoforms of OPA1 in mouse cells and tissues. This protocol can be applied to study changes in mitochondrial structure and function. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Western Blot Protocol for Isolating OPA1 Isoforms in Mouse Primary Skeletal Muscle Cells

Understanding the dynamic pathophysiology of diseases in the lung, such as asthma and chronic asthma, chronic obstructive pulmonary disease, and lung cancer, is crucial for the treatment, analysis, and outcome of these diseases. Unlike other traditional models, we suggest a protocol that is sustainable and reproducible and offers different analysis methods while maintaining in vivo lung architecture and immune dynamics. This protocol allows one to study the pathophysiological changes, including changes to the immune cells, cytokines, and mediators, in 30 precision-cut lung slices from a single murine lung. To accomplish this, the murine lung is infused with 2.5% low-melting-point agarose and is precision-cut-sliced. Our method also supports cell culture in refined medium and stimulation with clinically relevant stimuli, which helps to clarify the mechanisms of the disease. Evaluation of the samples and their supernatant includes multiplex assays, ELISA, histology, and immunohistochemistry. Additional sections are used to extract RNA for quantitative real-time PCR and RNA sequencing and/or other selected analysis, like flow cytometry. Using this method, we obtained murine lung slices that preserve the pathophysiology of the disease and allow a comprehensive analysis unlike other already-existing protocols. By retaining the dynamic immune mechanisms, we are able to see the histological damage caused by each disease. The results of this protocol can be used to improve our understanding and therapy options. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Obtaining precision-cut lung slices (PCLSs) from the murine lung

Support Protocol 1: LL/2 cell treatment with G-418 solution

Support Protocol 2: Murine model of lung adenocarcinoma and in vivo imaging

Support Protocol 3: H&E staining of PCLS sections

Support Protocol 4: Measuring cytokines by ELISA

Support Protocol 5: Measuring cytotoxic activity in PCLS conditioned medium

Support Protocol 6: Analysis of RNA from PCLSs by real-time PCR

Support Protocol 7: Flow cytometry analysis of cells isolated from PCLSs

Current Protocols is issuing corrections for the following protocol article.

Batenburg, K. L., Rohde, S. K., Cornelissen-Steijger, P., Breeuwsma, N., Heine, V. M., & Scheper, W. (2023). A human neuron/astrocyte co-culture to model seeded and spontaneous intraneuronal tau aggregation. Current Protocols, 3, e900. doi: 10.1002/cpz1.900

The current version online now includes these corrections and may be considered the authoritative version of record.