Bio-protocol最新文献

{"title":"Simple and Fail-safe Method to Transform Miniprep <i>Escherichia coli</i> Strain K12 Plasmid DNA Into Viable <i>Agrobacterium tumefaciens</i> EHA105 Cells for Plant Genetic Transformation.","authors":"Beenzu Siamalube, Emmanuel Ehinmitan, Maina Ngotho, Justus Onguso, Steven Runo","doi":"10.21769/BioProtoc.5174","DOIUrl":"10.21769/BioProtoc.5174","url":null,"abstract":"<p><p>Agrobacterium-mediated gene transformation method is a vital molecular biology technique employed to develop transgenic plants. Plants are genetically engineered to develop disease-free varieties, knock out unsettling traits for crop improvement, or incorporate an antigenic protein to make the plant a green factory for edible vaccines. The method's robustness was validated through successful transformations, demonstrating its effectiveness as a standard approach for researchers working in plant biotechnology. It enables the introduction of foreign DNA into plant genomes. Conventionally, plant genetic transformation has relied on time-consuming, costly, and technically demanding procedures, such as electroporation and chimeric viruses or biolistic methods, which usually yield variable transformation efficiencies. This study presents a simple and fail-safe protocol that involves a modified freeze-thaw and heat-shock concoction method. This approach involves a streamlined plasmid miniprep procedure to isolate high-quality plasmid DNA from <i>Escherichia coli</i> K12 strain, followed by a target-specific transfer into <i>A. tumefaciens</i> EHA105 strain. The optimized method minimizes DNA degradation and maximizes uptake by <i>Agrobacterium</i> cells, making it a reproducible and accessible protocol for various genetic engineering applications. The transformation efficiency is consistently high, enhancing plasmid uptake while maintaining cell viability, requiring minimal specialized equipment and reagents. The proposed protocol offers significant advantages, including simplicity, reliability, and cost-effectiveness, positioning it as a valuable alternative to traditional techniques in the field of plant biotechnology. Key features • Uses liquid nitrogen as a proxy for freezing. • Plasmid DNA from competent bacterial cells is extracted using a user-friendly high-copy isolation kit. • A maximum of five consecutive days is sufficient to complete the procedures.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 1","pages":"e5174"},"PeriodicalIF":1.0,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11717716/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142973879","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}

{"title":"Using HBmito Crimson to Observe Mitochondrial Cristae Through STED Microscopy.","authors":"Xichuan Ge, Wei Ren, Chunyan Shan, Peng Xi, Baoxiang Gao","doi":"10.21769/BioProtoc.5150","DOIUrl":"10.21769/BioProtoc.5150","url":null,"abstract":"<p><p>Mitochondrial cristae, formed by folding the mitochondrial inner membrane (IM), are essential for cellular energy supply. However, the observation of the IM is challenging due to the limitations in spatiotemporal resolution offered by conventional microscopy and the absence of suitable in vitro probes specifically targeting the IM. Here, we describe a detailed imaging protocol for the mitochondrial inner membrane using the Si-rhodamine dye HBmito Crimson, which has excellent photophysical properties, to label live cells for imaging via stimulated emission depletion (STED) microscopy. This allows for STED imaging over more than 500 frames (approximately one hour), with a spatial resolution of 40 nm, enabling the observation of cristae dynamics during various mitochondrial processes. The protocol includes detailed steps for cell staining, image acquisition, image processing, and resolution analysis. Utilizing the superior resolution of STED microscopy, the structure and complex dynamic changes of cristae can be visualized. Key features • The protocol is designed to visualize mitochondrial cristae in living cells using STED microscopy. • The protocol enables nanoscale observation of dynamic mitochondrial cristae. • Real-time observation of mitochondrial morphological changes, fusion, and fission events.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 1","pages":"e5150"},"PeriodicalIF":1.0,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11717718/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142973882","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}

{"title":"A Novel and Robust Method for Investigating Fungal Biofilm.","authors":"Biswambhar Biswas, Shumaiza Asif, Rekha Puria, Anil Thakur","doi":"10.21769/BioProtoc.5146","DOIUrl":"10.21769/BioProtoc.5146","url":null,"abstract":"<p><p><i>Candida auris</i>, labeled an urgent threat by the CDC, shows significant resilience to treatments and disinfectants via biofilm formation, complicating treatment/disease management. The inconsistencies in biofilm architecture observed across studies hinder the understanding of its role in pathogenesis. Our novel in vitro technique cultivates <i>C. auris</i> biofilms on gelatin-coated coverslips, reliably producing multilayer biofilms with extracellular polymeric substances (EPS). This method, applicable to other <i>Candida</i> species like <i>C. glabrata</i> and <i>C. albicans</i>, is cost-effective and mimics the niche of biofilm formation. It is suitable for high-throughput drug screening and repurposing efforts, aiding in the development of new therapeutics. Our technique represents a significant advancement in <i>Candida</i> biofilm research, addressing the need for consistent, reproducible biofilm models. We detail a step-by-step procedure for creating a substratum for biofilm growth and measuring biofilm thickness using confocal laser scanning microscopy (CLSM) and ultrastructure by scanning electron microscopy (SEM). This method provides consistent outcomes across various <i>Candida</i> species. Key features • The biofilm formed on gelatin surfaces mimics host conditions, replicating the multilayered structure and EPS, offering a more accurate model for studying <i>C. auris</i> biofilms. • This method is highly reproducible and suitable for drug screening and biofilm analysis through three-dimensional (3D) reconstruction. • This in vitro technique aids in studying biofilm formation, related virulence properties, and drug tolerance of <i>C. auris</i> and other <i>Candida</i> species. • The simple, cost-effective technique is ideal for screening novel inhibitors and repurposed drug libraries, facilitating the design/identification of new therapeutics against <i>Candida</i> species.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 1","pages":"e5146"},"PeriodicalIF":1.0,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11717715/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142973777","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}

{"title":"Microfluidic Cultures of Basal Forebrain Cholinergic Neurons for Assessing Retrograde Cell Death by Live Imaging.","authors":"Srestha Dasgupta, Mansi A Pandya, Wilma J Friedman","doi":"10.21769/BioProtoc.5149","DOIUrl":"10.21769/BioProtoc.5149","url":null,"abstract":"<p><p>Neurons are highly polarized cells, with axons that may innervate distant target regions. In the brain, basal forebrain cholinergic neurons (BFCNs) possess extensive axons that project to several target regions such as the cortex, hippocampus, and amygdala, and may be exposed to a specific microenvironment in their axon targets that may have retrograde effects on neuronal health. Interestingly, BFCNs express the pan-neurotrophin receptor p75NTR throughout life while also concomitantly co-expressing all Trk receptors, making them capable of responding to both mature and precursor neurotrophins to promote survival or apoptosis, respectively. Levels of these trophic factors may be modulated in the BFCN axon or soma microenvironment under neurodegenerative conditions such as seizure and brain injury. In this protocol, BFCNs are established in microfluidic devices for compartmental culture, with the aim of studying the effects of axon- or soma-specific stimulation of BFCNs for an in vitro representation of distal axon vs. soma environments as seen in vivo. This study further establishes a novel method of tracing and imaging live BFCNs exposed to stimuli in their distal axons with the aim of assessing retrograde cell death. The in vitro compartmental culture system of BFCNs that allows live imaging may be applied to investigate various effects of axon- or soma-specific stimuli that affect BFCN health, maintenance, and death, to model events that occur in the context of brain injury and neurodegenerative disorders. Key features • Separation of axons and soma of basal forebrain primary neurons in vitro using microfluidic chambers. • Compartmental/localized treatment of axons or somas of BFCNs. • Live imaging of retrogradely labeled BFCNs to assess cell death.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 1","pages":"e5149"},"PeriodicalIF":1.0,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11717710/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142973875","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}

{"title":"Cryopreservation Method for Preventing Freeze-Fracture of Small Muscle Samples.","authors":"Namrata Ghag, Joshua Tam, Rox R Anderson, Nashwa Cheema","doi":"10.21769/BioProtoc.5145","DOIUrl":"10.21769/BioProtoc.5145","url":null,"abstract":"<p><p>Histological techniques to study muscle are crucial for assessing skeletal muscle health. To preserve tissue morphology, samples are usually fixed in formaldehyde or cryopreserved immediately after excision from the body. Freezing samples in liquid nitrogen, using isopentane as a mediator for efficient cooling, preserves the tissue in its natural state. However, this method is highly susceptible to freeze-fracture artifacts, which alter or destroy tissue architecture. Isopentane is most commonly used in a semi-frozen/liquid state that is visually assessed by the experimenter, which can pose a challenge when freezing multiple tissues at a time or maintaining a consistent temperature. Furthermore, tissue size is also a confounding factor; depending on the size, freezing times can vary. In this study, we compare two different options for using isopentane while cryopreserving tissue. We also present an easy and reproducible method of freezing the soleus tissue of mice using frozen isopentane. This method decreased the occurrence of freeze-fractures by an order of magnitude, to ~4%, whereas the traditional method of cryopreservation resulted in ~56% freeze-fracturing. Key features • A uniform and highly reproducible protocol for freezing any tissue that is prone to freeze-fracture. • Removes the need to maintain a mixed state of isopentane. • Optimized cryopreservation method for the soleus muscle of mice. • Allows for prevention of peripheral freeze-fracture in tissue, which is the most susceptible region to freeze-fracture damage. Graphical overview.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 1","pages":"e5145"},"PeriodicalIF":1.0,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11717720/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142973871","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}

{"title":"Automatic Adaptive Algorithm for Delineation of Cerebral-Spinal Fluid Regions for Non-contrast Magnetic Resonance Imaging Volumetry and Cisternography in Mice.","authors":"Ryszard S Gomolka","doi":"10.21769/BioProtoc.5148","DOIUrl":"10.21769/BioProtoc.5148","url":null,"abstract":"<p><p>Magnetic resonance imaging (MRI) is an invaluable method of choice for anatomical and functional in vivo imaging of the brain. Still, accurate delineation of the brain structures remains a crucial task of MR image evaluation. This study presents a novel analytical algorithm developed in MATLAB for the automatic segmentation of cerebrospinal fluid (CSF) spaces in preclinical non-contrast MR images of the mouse brain. The algorithm employs adaptive thresholding and region growing to accurately and repeatably delineate CSF space regions in 3D constructive interference steady-state (3D-CISS) images acquired using a 9.4 Tesla MR system and a cryogenically cooled transmit/receive resonator. Key steps include computing a bounding box enclosing the brain parenchyma in three dimensions, applying an adaptive intensity threshold, and refining CSF regions independently in sagittal, axial, and coronal planes. In its original application, the algorithm provided objective and repeatable delineation of CSF regions in 3D-CISS images of sub-optimal signal-to-noise ratio, acquired with (33 μm)³ isometric voxel dimensions. It allowed revealing subtle differences in CSF volumes between aquaporin-4-null and wild-type littermate mice, showing robustness and reliability. Despite the increasing use of artificial neural networks in image analysis, this analytical approach provides robustness, especially when the dataset is insufficiently small and limited for training the network. By adjusting parameters, the algorithm is flexible for application in segmenting other types of anatomical structures or other types of 3D images. This automated method significantly reduces the time and effort compared to manual segmentation and offers higher repeatability, making it a valuable tool for preclinical and potentially clinical MRI applications. Key features • This protocol presents a fully automatic adaptive algorithm for the delineation of CSF space regions in 3D-CISS in vivo images of the mouse brain. • The algorithm represents an analytical method for adaptive CSF regions separation based on cumulative distribution of brain image intensities and contrast calculation-based slice-wise region growing. • Users can interactively alter the input parameters to modify the algorithm's output in a variety of 3D brain MR and μCT or CT images. • The algorithm is implemented in MATLAB 2021a and is compatible with all versions up to 2024a.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 1","pages":"e5148"},"PeriodicalIF":1.0,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11717714/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142973867","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}

{"title":"An Assay System for Plate-based Detection of Endogenous Peptide:<i>N</i>-glycanase/NGLY1 Activity Using A Fluorescence-based Probe.","authors":"Hiroto Hirayama, Tadashi Suzuki","doi":"10.21769/BioProtoc.5151","DOIUrl":"10.21769/BioProtoc.5151","url":null,"abstract":"<p><p>Cytosolic peptide:<i>N</i>-glycanase (PNGase/NGLY1 in mammals), an amidase classified under EC:3.5.1.52, is a highly conserved enzyme across eukaryotes that catalyzes the removal of <i>N</i>-glycans from glycoproteins, converting <i>N</i>-glycosylated asparagine residues into aspartic acid. This enzyme also plays a role in the quality control system for nascent glycoproteins. Despite the development of non-radioisotope-based assay systems such as those using <i>S</i>-alkylated RNase or fluorescent-labeled glycopeptides as substrates, these methods are incompatible with crude enzyme sources, primarily due to the degradation of reaction products by contaminating endogenous proteases. We previously developed an assay system using a 5-carboxyfluorescein-labeled glycosylated cyclo-heptapeptide (5FAM-GCP), a substrate remarkably resistant to endogenous peptidase activity. This system enables the accurate measurement of endogenous NGLY1 activity in various samples, including cell lines, tissues, peripheral blood mononuclear cells, and NGLY1-deficient patient-derived cells, without the interference of proteolytic degradation. We recently advanced this approach by producing a novel fluorescence resonance energy transfer (FRET)-based GCP probe (fGCP) and demonstrated its ability to detect endogenous NGLY1 activity across diverse enzyme sources via fluorescence on multiarray plates. This innovative and straightforward assay now offers reliable disease diagnostics and also allows the measurement of endogenous PNGase/NGLY1 activities across various organisms. Key features • fGCP assay enables measurement of endogenous PNGase/NGLY1 activity in cells and tissues. • An aliquot of 1-5 × 10⁶ cells or 50-100 μg of protein extract from tissues is used for this assay. • This assay enables microplate-based real-time measurement of endogenous PNGase/NGLY1 activities. • This protocol requires a fluorescence plate reader equipped with an incubation function.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 1","pages":"e5151"},"PeriodicalIF":1.0,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11717711/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142973786","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}

{"title":"Temporally and Spatially Controlled Age-Related Prostate Cancer Model in Mice.","authors":"Sen Liu, Keyi Shen, Zixuan Li, Seleste Rivero, Qiuyang Zhang","doi":"10.21769/BioProtoc.5144","DOIUrl":"10.21769/BioProtoc.5144","url":null,"abstract":"<p><p>The initiation and progression of prostate cancer (PCa) are associated with aging. In the history of age-related PCa research, mice have become a more popular animal model option than any other species due to their short lifespan and rapid reproduction. However, PCa in mice is usually induced at a relatively young age, while it spontaneously develops in humans at an older age. Thus, it is essential to develop a method by which the PCa initiation and progression timeline can be strictly controlled to mimic human physiological conditions. One milestone in this field was the identification of the prostate-specific transcription factor, Probasin (Pb), which allowed for the prostate-specific expression of genes knocked into the mice's genome. Another milestone is the establishment of the preclinical mouse model with <i>Pten</i> conditionally knocked out in the prostate tissue, which closely mimics the formation and growth of human PCa. Hereby, we present the prostate-specific temporally and spatially controlled <i>Pten</i> knockout PCa mouse model that can be induced using an adenovirus-based Cre-LoxP system. The Cre recombinase (Cre) is inserted into an adenovirus vector. Unlike Pb-Cre knock-in models (which are spatially but not temporally controlled), the expression of Cre is activated to knock out <i>Pten</i> from the mice's prostate epithelial cells once injected. The viral delivery procedures strictly control the location and time of <i>Pten</i> knockout. This novel approach provides a powerful age-related murine model for PCa, emphasizing the effect of aging on prostate carcinogenesis. Key features • In vivo delivery of Cre recombinase adenovirus (Ad-Cre-Luc) in <i>Pten</i> LoxP/LoxP (L/L) mice. • Generation of Cre-expressing Ad-Cre-Luc-mediated ablation of <i>Pten</i> in anterior prostate epithelial cells of adult <i>Pten</i> L/L mice at different ages. • The Ad-Cre-Luc-mediated ablation of <i>Pten</i> leads to hyperplasia that progresses through prostatic intraepithelial neoplasia (PIN) to adenocarcinoma. • PIN refers to the non-cancerous growth of epithelial cells in the prostate tissue-not cancer but a precursor of prostate cancer [1].</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 1","pages":"e5144"},"PeriodicalIF":1.0,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11717534/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142973881","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}

{"title":"Capacitance Measurements of Exocytosis From AII Amacrine Cells in Retinal Slices.","authors":"Espen Hartveit, Margaret L Veruki","doi":"10.21769/BioProtoc.5147","DOIUrl":"10.21769/BioProtoc.5147","url":null,"abstract":"<p><p>During neuronal synaptic transmission, the exocytotic release of neurotransmitters from synaptic vesicles in the presynaptic neuron evokes a change in conductance for one or more types of ligand-gated ion channels in the postsynaptic neuron. The standard method of investigation uses electrophysiological recordings of the postsynaptic response. However, electrophysiological recordings can directly quantify the presynaptic release of neurotransmitters with high temporal resolution by measuring the membrane capacitance before and after exocytosis, as fusion of the membrane of presynaptic vesicles with the plasma membrane increases the total capacitance. While the standard technique for capacitance measurement assumes that the presynaptic cell is unbranched and can be represented as a simple resistance-capacitance (RC) circuit, neuronal exocytosis typically occurs at a distance from the soma. Even in such cases, however, it can be possible to detect a depolarization-evoked increase in capacitance. Here, we provide a detailed, step-by-step protocol that describes how \"Sine + DC\" (direct current) capacitance measurements can quantify the exocytotic release of neurotransmitters from AII amacrine cells in rat retinal slices. The AII is an important inhibitory interneuron of the mammalian retina that plays an important role in integrating rod and cone pathway signals. AII amacrines release glycine from their presynaptic dendrites, and capacitance measurements have been important for understanding the release properties of these dendrites. When the goal is to directly quantify the presynaptic release, there is currently no other competing method available. This protocol includes procedures for measuring depolarization-evoked exocytosis, using both standard square-wave pulses, arbitrary stimulus waveforms, and synaptic input. Key features • Quantification of exocytosis with the Sine + DC technique for visually targeted AII amacrines in retinal slices, using voltage-clamp and whole-cell patch-clamp recording. • Because exocytosis occurs away from the somatic recording electrode, the sine wave frequency must be lower than for the standard Sine + DC technique. • Because AII amacrines are electrically coupled, the sine wave frequency must be sufficiently high to avoid interference from other cells in the electrically coupled network. • The protocol includes procedures for measuring depolarization-evoked exocytosis using standard square-wave pulses, stimulation with arbitrary and prerecorded stimulus waveforms, and activation of synaptic inputs.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 1","pages":"e5147"},"PeriodicalIF":1.0,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11717719/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142973870","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}

{"title":"An Open-source Python Tool for Traction Force Microscopy on Micropatterned Substrates.","authors":"Artur Ruppel, Vladimir Misiak, Martial Balland","doi":"10.21769/BioProtoc.5156","DOIUrl":"10.21769/BioProtoc.5156","url":null,"abstract":"<p><p>Cell-generated forces play a critical role in driving and regulating complex biological processes, such as cell migration and division and cell and tissue morphogenesis in development and disease. Traction force microscopy (TFM) is an established technique developed in the field of mechanobiology used to quantify cellular forces exerted on soft substrates and internal mechanical tissue stresses. TFM measures cell-generated traction forces in 2D or 3D environments with varying mechanical and biochemical properties. This technique involves embedding fiducial markers in the substrate, imaging substrate deformations caused by the cells, and using mathematical models to infer forces. This protocol compiles procedures from various previously published studies and software packages and describes how to perform TFM on 2D micropatterned substrates. Although not the focus of this protocol, the methods and software packages shown here also allow to perform monolayer stress microscopy (MSM), a method to calculate internal mechanical stress within the cells by modeling them as a thin plate with linear and homogeneous material properties. TFM and MSM are non-invasive methods capable of yielding spatially and temporally resolved force and stress maps with high throughput. As such, they enable the generation of rich datasets, which can provide valuable insights into the roles of cell-generated forces in various physiological and pathological processes. Key features • TFM and MSM protocol for 2D micropatterned polyacrylamide substrates, from sample preparation over imaging to data analysis with provided code. • Sample preparation method is based on Tseng et al. [1]. • TFM analysis is done with Python custom code and is optimized for batch analysis of movies. • MSM analysis is done with pyTFM from Bauer et al. [2].</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 1","pages":"e5156"},"PeriodicalIF":1.0,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11717713/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142973863","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}