Methods and Protocols最新文献_第2页

A Scalable Method to Fabricate 2D Hydrogel Substrates for Mechanobiology Studies with Independent Tuning of Adhesiveness and Stiffness. 为机械生物学研究制造二维水凝胶基底的可扩展方法，可独立调节粘附性和硬度。

IF 2.3

Methods and Protocols Pub Date : 2024-09-26 DOI: 10.3390/mps7050075

Alessandro Gandin, Veronica Torresan, Tito Panciera, Giovanna Brusatin

{"title":"A Scalable Method to Fabricate 2D Hydrogel Substrates for Mechanobiology Studies with Independent Tuning of Adhesiveness and Stiffness.","authors":"Alessandro Gandin, Veronica Torresan, Tito Panciera, Giovanna Brusatin","doi":"10.3390/mps7050075","DOIUrl":"https://doi.org/10.3390/mps7050075","url":null,"abstract":"Mechanical signals from the extracellular matrix are crucial in guiding cellular behavior. Two-dimensional hydrogel substrates for cell cultures serve as exceptional tools for mechanobiology studies because they mimic the biomechanical and adhesive characteristics of natural environments. However, the interdisciplinary knowledge required to synthetize and manipulate these biomaterials typically restricts their widespread use in biological laboratories, which may not have the material science expertise or specialized instrumentation. To address this, we propose a scalable method that requires minimal setup to produce 2D hydrogel substrates with independent modulation of the rigidity and adhesiveness within the range typical of natural tissues. In this method, norbornene-terminated 8-arm polyethylene glycol is stoichiometrically functionalized with RGD peptides and crosslinked with a di-cysteine terminated peptide via a thiol-ene click reaction. Since the synthesis process significantly influences the final properties of the hydrogels, we provide a detailed description of the chemical procedure to ensure reproducibility and high throughput results. We demonstrate examples of cell mechanosignaling by monitoring the activation state of the mechanoeffector proteins YAP/TAZ. This method effectively dissects the influence of biophysical and adhesive cues on cell behavior. We believe that our procedure will be easily adopted by other cell biology laboratories, improving its accessibility and practical application.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 5","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11510107/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504287","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Co-Culture of Gut Bacteria and Metabolite Extraction Using Fast Vacuum Filtration and Centrifugation. 利用快速真空过滤和离心法联合培养肠道细菌并提取代谢物

IF 2.3

Methods and Protocols Pub Date : 2024-09-19 DOI: 10.3390/mps7050074

Asha Guraka, Richard Duff, Joe Waldron, Gyanendra Tripathi, Ali Kermanizadeh

{"title":"Co-Culture of Gut Bacteria and Metabolite Extraction Using Fast Vacuum Filtration and Centrifugation.","authors":"Asha Guraka, Richard Duff, Joe Waldron, Gyanendra Tripathi, Ali Kermanizadeh","doi":"10.3390/mps7050074","DOIUrl":"10.3390/mps7050074","url":null,"abstract":"This protocol describes a robust method for the extraction of intra and extracellular metabolites of gut bacterial mono and co-cultures. In recent years, the co-culture techniques employed in the field of microbiology have demonstrated significant importance in regard to understanding cell-cell interactions, cross-feeding, and the metabolic interactions between different bacteria, fungi, and microbial consortia which enable the mimicking of complex co-habitant conditions. This protocol highlights a robust reproducible physiologically relevant culture and extraction protocol for the co-culture of gut bacterium. The novel extraction steps are conducted without using quenching and cell disruption through bead-cell methods, freeze-thaw cycles, and sonication, which tend to affect the physical and biochemical properties of intracellular metabolites and secretome. The extraction procedure of inoculated bacterial co-cultures and monocultures use fast vacuum filtration and centrifugation. The extraction methodology is fast, effective, and robust, requiring 4 h to complete.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 5","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11417889/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291449","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Microneedle System Coated with Hydrogels Containing Protoporphyrin IX for Potential Application in Pharmaceutical Technology. 涂有原卟啉 IX 水凝胶的微针系统在制药技术中的潜在应用。

IF 2.3

Methods and Protocols Pub Date : 2024-09-13 DOI: 10.3390/mps7050073

Beata Czarczynska-Goslinska, Tomasz Goslinski, Agata Roszak, Anna Froelich, Piotr Szyk, Dariusz T Mlynarczyk, Lukasz Sobotta, Irena Budnik, Oliwia Kordyl, Tomasz Osmałek

{"title":"Microneedle System Coated with Hydrogels Containing Protoporphyrin IX for Potential Application in Pharmaceutical Technology.","authors":"Beata Czarczynska-Goslinska, Tomasz Goslinski, Agata Roszak, Anna Froelich, Piotr Szyk, Dariusz T Mlynarczyk, Lukasz Sobotta, Irena Budnik, Oliwia Kordyl, Tomasz Osmałek","doi":"10.3390/mps7050073","DOIUrl":"10.3390/mps7050073","url":null,"abstract":"The article aims to outline the potential of treating malignant skin cancer with microneedles covered with polymer layers containing a photosensitizer-protoporphyrin IX disodium salt (PPIX). The usefulness of stereolithography (SLA), which is a form of 3D-printing technology, for the preparation of a microneedle system with protoporphyrin IX was demonstrated. The SLA method allowed for pyramid-shaped microneedles to be printed that were covered with three different 0.1% PPIX hydrogels based on sodium alginate, xanthan, and poloxamer. Rheological tests and microscopic analysis of the hydrogels were performed. Microneedles coated with two layers of poloxamer-based hydrogel containing 0.1% PPIX were subjected to release tests in Franz diffusion cells. The release profile of PPIX initially increased and then remained relatively constant. The amount of substance released after a four-hour test in three Franz cells was 0.2569 ± 0.0683 mg/cm2. Moreover, the acute toxicity of this type of microneedle was assessed using the Microtox system. The obtained results show the usefulness of further development studies on microneedles as carriers of photosensitizing agents.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 5","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11417702/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291453","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Generating Cytokines and Growth Factors for Functional Activity: Feasibility of Method Using MIF Protein. 产生细胞因子和生长因子的功能活性：使用 MIF 蛋白的方法的可行性。

IF 2.3

Methods and Protocols Pub Date : 2024-09-12 DOI: 10.3390/mps7050072

Hiba Osmani, Ishrya Sharma, Shannon Moonah

{"title":"Generating Cytokines and Growth Factors for Functional Activity: Feasibility of Method Using MIF Protein.","authors":"Hiba Osmani, Ishrya Sharma, Shannon Moonah","doi":"10.3390/mps7050072","DOIUrl":"10.3390/mps7050072","url":null,"abstract":"Cytokines and growth factors are signaling molecules that regulate a variety of biological processes. Understanding their role is essential for basic research and clinical utilization. Thus, cytokines and growth factors are widely used throughout research labs in a significant number of applications. Additionally, genetic polymorphisms result in variant forms of cytokines and growth factors, which can alter their function. Becoming more common, researchers will need to generate these important proteins and their variants themselves in functional forms for activity studies. The expression systems used to generate these proteins can have a major impact on their function. In some instances, post-translational modifications are needed to produce a functionally active protein, which can only be conducted using eukaryotic expression systems. Ideally, for functional relevance, a human expression system should be used for human-related research and applications. Most human cell-based expression systems primarily use HEK (Human Embryonic Kidney) cells; however, relying on just one cell type can lead to several issues, considering the variety of proteins derived from various cell sources. Here, we provide a protocol to effectively and efficiently generate functional recombinant proteins, taking into consideration the diverse range of proteins from different cell types throughout the human body.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 5","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11417903/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291452","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

FAMS-A Targeted Fatty Acid Mass Spectrometry Method for Monitoring Free Fatty Acids from Polysorbate Hydrolysis. FAMS- 用于监测聚山梨醇酯水解游离脂肪酸的定向脂肪酸质谱法。

IF 2.3

Methods and Protocols Pub Date : 2024-09-07 DOI: 10.3390/mps7050071

Anja Bathke, Sina Hoelterhoff, Jan Wendler, Inn H Yuk, Christian H Bell

{"title":"FAMS-A Targeted Fatty Acid Mass Spectrometry Method for Monitoring Free Fatty Acids from Polysorbate Hydrolysis.","authors":"Anja Bathke, Sina Hoelterhoff, Jan Wendler, Inn H Yuk, Christian H Bell","doi":"10.3390/mps7050071","DOIUrl":"10.3390/mps7050071","url":null,"abstract":"Polysorbates are the predominant surfactants used to stabilize protein formulations. Unfortunately, polysorbates can undergo hydrolytic degradation, which releases fatty acids that can accumulate to form visible particles. The detection and quantitation of these fatty acid degradation products are critical for assessing the extent of polysorbate degradation and the associated risks of particle formation. We previously developed a user-friendly mass spectrometric method called Fatty Acids by Mass Spectrometry (FAMS) to quantify the free fatty acids. The FAMS method was validated according to ICH Q2 (R1) guidelines and is suitable for a wide range of products, buffers and protein concentrations. The end-to-end workflow can be automated from sample preparation to data analysis. To broaden method accessibility, the QDa detector selected for fatty acid measurement does not require specific mass spectrometry experience. We provide here a detailed procedure for both manual and automated sample preparation for high-throughput analysis. In addition, we highlight in this protocol the critical operational details, procedural watchouts and troubleshooting tips to support the successful execution of this method in another laboratory.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 5","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11417882/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291451","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The Burden of Sleep/Wake Disorders: Excessive Daytime Sleepiness and Insomnia Project. 睡眠/觉醒障碍的负担：白天过度嗜睡和失眠项目。

IF 2.3

Methods and Protocols Pub Date : 2024-09-06 DOI: 10.3390/mps7050070

Marina Tüzün, Ulf Kallweit, Stefan Seidel, Olga Endrich, Sven Trelle, Maurizio A Leone, Oliviero Bruni, Richard Dodel, Maria Konti, Maria Lolich, Elisabetta Pupillo, Dauren Ramankulov, Luca Vignatelli, Carla Meyer-Massetti, Markus Schmidt, Claudio L A Bassetti

{"title":"The Burden of Sleep/Wake Disorders: Excessive Daytime Sleepiness and Insomnia Project.","authors":"Marina Tüzün, Ulf Kallweit, Stefan Seidel, Olga Endrich, Sven Trelle, Maurizio A Leone, Oliviero Bruni, Richard Dodel, Maria Konti, Maria Lolich, Elisabetta Pupillo, Dauren Ramankulov, Luca Vignatelli, Carla Meyer-Massetti, Markus Schmidt, Claudio L A Bassetti","doi":"10.3390/mps7050070","DOIUrl":"10.3390/mps7050070","url":null,"abstract":"Excessive daytime sleepiness (EDS) and insomnia (IN) complaints represent the most common sleep/wake disorders. Currently, the specific needs of these patients and their relatives, as well as the overall socio-economic burden of IN and EDS remains widely unexplored. This pilot study to be carried out in Switzerland is a retro- and prospective, national, one-center cohort observational study for the systematic evaluation of the burden of EDS and IN and its evolution 12 months after the first assessment. Patient recruitment will be organized through 7-8 primary care providers (primary/general care practitioners and pharmacies). Primary outcomes are the prevalence of EDS/IN in the primary care setting and the association between EDS/IN with health-related quality of life (QOL) as assessed with the established instruments. Secondary outcomes are the association between EDS/IN with the presence of comorbidities, number of injuries/accidents, and number of sick/leave days for the subgroup of working subjects. Calculation of direct per-patient costs will be undertaken to analyze the economic implications of sleep/wake disorders, providing valuable insights into the financial burden experienced by affected individuals within the healthcare system. This research will provide information on the feasibility of such a study and inform on aspects of the QOL most associated with EDS/IN. Based on this pilot project, a European multicenter study on the burden of sleep/wake disorders will be conducted by the European Academy of Neurology.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 5","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11417944/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291456","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Optimization of Methodologies to Study Freeze/Thaw Processes in Drug Substance Bottles. 优化研究药物瓶中冻融过程的方法。

IF 2.3

Methods and Protocols Pub Date : 2024-09-04 DOI: 10.3390/mps7050068

Sarah S Peláez, Hanns-Christian Mahler, Jörg Huwyler, Andrea Allmendinger

{"title":"Optimization of Methodologies to Study Freeze/Thaw Processes in Drug Substance Bottles.","authors":"Sarah S Peláez, Hanns-Christian Mahler, Jörg Huwyler, Andrea Allmendinger","doi":"10.3390/mps7050068","DOIUrl":"10.3390/mps7050068","url":null,"abstract":"Biological drug substance (DS) is often frozen to enhance storage stability, prolong shelf life, and increase flexibility during manufacturing. However, the freezing and thawing (F/T) of bulk DS at the manufacturing scale can impact product quality as a result of various critical conditions, including cryo-concentration during freezing, which are influenced, among other things, by product-independent process parameters (e.g., container type, fill level, F/T equipment, and protocols). In this article, we report the optimization of two major methodologies to study product-independent process parameters in DS bottles at the manufacturing scale, namely the recording of temperature profiles and liquid sampling after thawing to quantify the concentration gradients in the solution. We report experimentally justified measuring positions for temperature recordings, especially for the selection of the last point to freeze position, and highlight the implementation of camera-assisted inspection to determine the last point to thaw and the actual thawing time. In particular, we provide, for the first time, a detailed description of the technical implementation of these two measuring set-ups. Based on the reported case studies, we recommend choosing relevant measuring positions as a result of initial equipment characterization, resulting in a resource-conscious study set-up.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 5","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11417747/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291455","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

CO₂-Free On-Stage Incubator for Live Cell Imaging of Cholangiocarcinoma Cell Migration on Microfluidic Device. 用于胆管癌细胞在微流控装置上迁移的活细胞成像的无二氧化碳阶段培养箱

IF 2.3

Methods and Protocols Pub Date : 2024-09-04 DOI: 10.3390/mps7050069

Shahab Ud Din, Puey Ounjai, Arthit Chairoungdua, Werasak Surareungchai

{"title":"CO2-Free On-Stage Incubator for Live Cell Imaging of Cholangiocarcinoma Cell Migration on Microfluidic Device.","authors":"Shahab Ud Din, Puey Ounjai, Arthit Chairoungdua, Werasak Surareungchai","doi":"10.3390/mps7050069","DOIUrl":"10.3390/mps7050069","url":null,"abstract":"Long-term live cell imaging requires sophisticated and fully automated commercial-stage incubators equipped with specified inverted microscopes to regulate temperature, CO2 content, and humidity. In this study, we present a CO2-free on-stage incubator specifically designed for use across various cell culture platforms, enabling live cell imaging applications. A simple and transparent incubator was fabricated from acrylic sheets to be easily placed on the stages of most inverted microscopes. We successfully performed live-cell imaging of cholangiocarcinoma (CCA) cells and HeLa cell dynamics in both 2D and 3D microenvironments over three days. We also analyzed directed cell migration under high serum induction within a microfluidic device. Interesting phenomena such as \"whole-colony migration\", \"novel type of collective cell migration\" and \"colony formation during cell and colony migration\" are reported here for the first time, to the best of our knowledge. These phenomena may improve our understanding of the nature of cell migration and cancer metastasis.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 5","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11417791/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291448","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Comparison of Antigen Retrieval Methods for Immunohistochemical Analysis of Cartilage Matrix Glycoproteins Using Cartilage Intermediate Layer Protein 2 (CILP-2) as an Example. 以软骨中间层蛋白 2 (CILP-2) 为例，比较用于软骨基质糖蛋白免疫组化分析的抗原检索方法。

IF 2.3

Methods and Protocols Pub Date : 2024-08-24 DOI: 10.3390/mps7050067

Taavi Torga, Siim Suutre, Kalle Kisand, Marina Aunapuu, Andres Arend

{"title":"Comparison of Antigen Retrieval Methods for Immunohistochemical Analysis of Cartilage Matrix Glycoproteins Using Cartilage Intermediate Layer Protein 2 (CILP-2) as an Example.","authors":"Taavi Torga, Siim Suutre, Kalle Kisand, Marina Aunapuu, Andres Arend","doi":"10.3390/mps7050067","DOIUrl":"10.3390/mps7050067","url":null,"abstract":"The aim of this study was to compare different antigen retrieval methods to improve the outcome of immunohistochemistry (IHC) performed on osteoarthritic (OA) cartilage obtained from total knee replacement operation. A voluminous and dense extracellular matrix of articular cartilage inhibits antibody penetration, and therefore, proteins present at low concentrations and masked during fixation may need antigen retrieval to enhance an IHC outcome. We focused on the IHC detection of a minor but diagnostically promising cartilage glycoprotein, CILP-2 (cartilage intermediate layer protein 2), to demonstrate the effect of four different protocols: (1) heat-induced epitope retrieval (HIER), (2) proteolytic-induced epitope retrieval applying proteinase K and hyaluronidase (PIER), (3) HIER combined with PIER, and (4) no antigen retrieval (control). A semi-quantitative staining assessment based on the CILP-2 staining extent was applied. Out of the tested antigen retrieval protocols, the best CILP-2 IHC staining results were achieved by PIER. Combining PIER with HIER did not improve CILP-2 staining in the given experimental setting. Rather the opposite, the application of heat reduced the positive effect of PIER on CILP-2 staining and resulted in the frequent detachment of sections from the slides. Our findings emphasize the need for proper adaptation of antigen retrieval protocols for IHC to maximize the quantitative evaluation of minor matrix proteins in OA articular cartilage samples.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 5","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-08-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11417793/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291450","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Nanoparticle Lysis of Cryptosporidium Oocysts. 纳米粒子溶解隐孢子虫卵囊。

IF 2.3

Methods and Protocols Pub Date : 2024-08-23 DOI: 10.3390/mps7050066

Ameya Vaidya, Claire Bankier, Helinor Johnston, Helen Bridle

引用次数: 0