Bio-protocol最新文献

{"title":"Single-Particle Tracking of AMPA Receptor-Containing Vesicles.","authors":"Victor C Wong, Deepika Walpita, Zhe J Liu, Erin K O'Shea","doi":"10.21769/BioProtoc.5325","DOIUrl":"10.21769/BioProtoc.5325","url":null,"abstract":"<p><p>AMPA-type receptors are transported large distances to support synaptic plasticity at distal dendritic locations. Studying the motion of AMPA receptor⁺ vesicles can improve our understanding of the mechanisms that underlie learning and memory. Nevertheless, technical challenges that prevent the visualization of AMPA receptor⁺ vesicles limit our ability to study how these vesicles are trafficked. Existing methods rely on the overexpression of fluorescent protein-tagged AMPA receptors from plasmids, resulting in a saturated signal that obscures vesicles. Photobleaching must be applied to detect individual AMPA receptor⁺ vesicles, which may eliminate important vesicle populations from analysis. Here, we present a protocol to study AMPA receptor⁺ vesicles that addresses these challenges by 1) tagging AMPA receptors expressed from native loci with HaloTag and 2) employing a block-and-chase strategy with Janelia Fluor-conjugated HaloTag ligand to achieve sparse AMPA receptor labeling that obviates the need for photobleaching. After timelapse imaging is performed, AMPA receptor⁺ vesicles can be identified during image analysis, and their motion can be characterized using a single-particle tracking pipeline. Key features • Track and characterize the motion of AMPAR GluA1⁺ vesicles in cultured rat hippocampal neurons. • GluA1 tagged with HaloTag (GluA1-HT) is expressed from native <i>Gria1</i> loci to avoid overexpression. • Sparse GluA1-HT labeling densities can be achieved without photobleaching via a block-and-chase strategy that utilizes Janelia Fluor (JF) dyes conjugated to HaloTag ligand (HTL). • GluA1-HT⁺ vesicles are identified during image analysis, and their motion is characterized using single-particle tracking (SPT) and hidden Markov modeling with Bayesian model selection (HMM-Bayes).</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 11","pages":"e5325"},"PeriodicalIF":1.0,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12177357/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144334542","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":"Silencing Arbuscular Mycorrhizal Fungal Gene Using Chitosan Nanoparticle-Mediated dsRNA Delivery System.","authors":"Chumei Yan, Yuemin Wang, Qianhuang Guo, Hongjuan Huan, Siwei Wang, Xiaoning Fan, Xianan Xie","doi":"10.21769/BioProtoc.5326","DOIUrl":"10.21769/BioProtoc.5326","url":null,"abstract":"<p><p>It has been discovered that many phytopathogenic fungi can absorb exogenous double-stranded RNAs (dsRNAs) to silence target genes, inhibiting fungal growth and pathogenicity for plant protection. In our recent report, the beneficial arbuscular mycorrhizal (AM) fungi are capable of acquiring external naked dsRNAs; however, whether the dsRNAs can be delivered into AM fungi through nanocarriers remains to be investigated. Here, we introduce a simple and advanced method for in vitro synthesizing chitosan (CS)/dsRNA polyplex nanoparticles (PNs) to silence the target gene in the AM fungus <i>Rhizophagus irregularis</i>. This method is straightforward, requiring minimal modifications, and is both efficient and eco-friendly, offering potential for rapid application in elucidating gene functions in AM fungi. Key features • The chitosan can carry the dsRNA derived from the AM fungus <i>Rhizophagus irregularis</i>. • CS/dsRNA polyplex nanoparticles (PNs) can successfully silence the target gene in the AM fungus <i>R. irregularis</i>. • CS/dsRNA PNs can be applied to the characterization of AM fungal genes via the spray-induced gene silencing (SIGS) approach. • This protocol can be applied in asymbiotic and symbiotic cultures of AM fungi. Graphical overview Overview of the chitosan/dsRNA gene silencing procedures.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 11","pages":"e5326"},"PeriodicalIF":1.0,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12152106/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144287566","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":"In-house Fabrication of Nanoplastics of Tunable Composition and Application: Assessment of Bioelectric Changes in Primary Rat Lung Alveolar Epithelial Cell Monolayers Exposed to Nanoplastics.","authors":"Ricki Chairil, Juan R Alvarez, Arnold Sipos, Noah Malmstadt, Edward D Crandall, Kwang-Jin Kim","doi":"10.21769/BioProtoc.5329","DOIUrl":"10.21769/BioProtoc.5329","url":null,"abstract":"<p><p>Plastic pollution presents a looming danger to the environment and virtually all life on planet Earth. Especially pernicious are nanoplastics (NPs), which are plastic fragments with dimensions ≤1 μm. Conventional detection methods are ineffective for NPs, while their high specific surface area renders them efficient carriers of toxic substances; additionally, they may even be inherently toxic. Although NP waste chiefly arises from environmental weathering of larger plastic fragments, most published studies employed manufactured pristine NPs of uniform size and shape. Furthermore, almost all NP effects were studied using polystyrene (PS) as a convenient model material, despite PS accounting for <6% of all plastic pollution. There is thus an urgent need to expand investigations of environmental NP pollution and effects on biota. The present work provides a comprehensive roadmap for studying the effects of \"real-world\" NP pollution on living systems, using, for example, lung alveolar epithelial cells on which such NPs deposit by breathing ambient air. Herein, we describe detailed in-house methods to fabricate various NPs that are weathered with UV light and O₃ gas exposure to more closely mimic real environmental NPs. We also illustrate a simple and straightforward bioelectrical method for assessing passive and active ion transport properties of primary rat lung alveolar epithelial cell monolayers as a model for the distal mammalian lung exposed to one of the generated NPs. This protocol allows researchers to rapidly and more accurately assess the biological impact of various simulated environmental NPs on a vulnerable air-blood barrier in the lung. Key features • Many simulated weathered environmental NPs can be produced at high concentrations (up to 120 mg/mL) and yields (up to 12 mg/g bulk plastic). • Any plastic waste can be \"nano-sized\" with this protocol and then studied for impacts on active and passive ion transport properties of cell monolayer models. • Methods described herein are very relevant for studying environmental pollution effects, since NPs are found in many different shapes, sizes, and compositions. • NP weathering and generation methods do not require any expensive or specialty lab instruments.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 11","pages":"e5329"},"PeriodicalIF":1.0,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12152108/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144287547","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":"Quantification of Folded and Misfolded Proinsulin Forms Using Nonreducing SDS-PAGE and Proinsulin-Specific Immunoblotting.","authors":"Anoop Arunagiri, Insook Jang, Pamela Itkin-Ansari, Randal J Kaufman, Peter Arvan","doi":"10.21769/BioProtoc.5337","DOIUrl":"10.21769/BioProtoc.5337","url":null,"abstract":"<p><p>We have observed that some proinsulin molecules in pancreatic islets and beta cell lines have incomplete or improper intramolecular disulfide bonds. These misfolded monomers can form intermolecular disulfide-linked complexes. Accurately quantifying the fraction of proinsulin molecules contained in these complexes is challenging. By proinsulin immunoblotting after nonreducing SDS-PAGE, the signal for disulfide-linked complexes can exceed the total proinsulin signal detected after reducing SDS-PAGE (i.e., overestimating the abundance of misfolded species due to antibody affinity differences). However, after modification of the SDS-PAGE and electrotransfer protocol, we have been able to more accurately estimate the fraction of proinsulin monomers folded to the native state, as well as misfolded proinsulin monomers and disulfide-linked complexes. Our improved technique offers the ability to obtain a more precise assessment of proinsulin misfolding under different environmental conditions in beta cells and normal islets and in diabetes. Key features • The protocol introduces a modification of a technique that enables clear separation and accurate quantification of native and non-native proinsulin monomers and aberrant disulfide-linked oligomers. • The protocol requires modifications to the standard SDS-PAGE and electrotransfer protocol to address quantitation inaccuracies that result from variations in antibody affinity. • Side-by-side comparison demonstrates that the standard immunoblotting method underestimates proinsulin monomers and overestimates the abundance of proinsulin disulfide-linked complexes. • This method is applicable to the study of recombinant proinsulin in heterologous cells, pancreatic β-cell lines, rodent or human pancreatic islets, and human iPSCs.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 11","pages":"e5337"},"PeriodicalIF":1.0,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12152102/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144287565","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":"Proliferation Assay Using Cryopreserved Porcine Peripheral Mononuclear Cells Stimulated With Concanavalin A and Analyzed With FCS Express^TM 7.18 Software.","authors":"Marlene Bravo-Parra, Rahul K Nelli, Lu Yen, Gino Castillo, Juan Carlos Mora-Díaz, Ana F Castillo-Espinoza, Luis G Giménez-Lirola","doi":"10.21769/BioProtoc.5332","DOIUrl":"10.21769/BioProtoc.5332","url":null,"abstract":"<p><p>In vitro lymphocyte proliferation assays are essential for assessing immune responses and antiproliferative drug efficacy. Such assays rely on antigen presentation or mitogen stimulation, with performance determined by reagent concentration and incubation time. Although splenocytes are often used, peripheral blood mononuclear cells (PBMCs) offer more accessible and practical sampling. However, a streamlined protocol for porcine PBMCs proliferation with robust batch analysis has been lacking. We therefore developed a detailed workflow for inducing proliferation in cryopreserved porcine PBMCs using 5 μg/mL concanavalin A (ConA). The protocol covers cell isolation, cryopreservation, ConA stimulation, CD4+ T-cell staining, flow cytometry acquisition and gating on an Attune NxT instrument, and batch analysis with FCS Express^TM 7.18. This approach yielded 78.9% viable cells, of which 33.8% were CD4+ lymphocytes. Moreover, 93.9% (n = 216) of cells proliferated, yielding up to nine cell generations. Batch analysis in FCS Express^TM enhanced the accuracy and interpretation of proliferation metrics. This validated protocol provides a reliable framework for generating consistent proliferation data in porcine immunology studies. Key features • Optimized proliferation assay for cryopreserved porcine PBMCs: Ideal for immunological studies and biomedical research using swine models. • Precise acquisition and gating with Attune NxT flow cytometer: Ensures accurate identification and analysis of CD4+ T lymphocytes. • Robust data analysis using FCS Express^TM 7.18 software: Facilitates reliable interpretation of lymphocyte proliferation and division indexes. • Efficient 4.5-day protocol: Enables timely and reproducible experiments in clinical and research settings involving porcine immune cells. Graphical overview Proliferation assay process from cryopreserved porcine peripheral blood mononuclear cells (PBMCs).</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 11","pages":"e5332"},"PeriodicalIF":1.0,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12152109/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144287552","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":"General Maintenance and Reactivation of iSLK Cell Lines.","authors":"Ariana C Calderón-Zavala, Aaron S Mendez, Ekaterina E Heldwein","doi":"10.21769/BioProtoc.5334","DOIUrl":"10.21769/BioProtoc.5334","url":null,"abstract":"<p><p>Since the establishment of the iSLK-BAC16 cell culture system, iSLK-BAC16 cells and their derivatives have been widely used for Kaposi's sarcoma-associated herpesvirus (KSHV) studies. However, iSLK-BAC16 cells can be difficult to work with, in part due to the lack of standardized protocols and conflicting troubleshooting suggestions. Here, we describe the protocol for general iSLK-BAC16 cell culture and reactivation, which induces lytic KSHV replication and virion production. This protocol achieves robust levels of KSHV reactivation in our hands and can be readily used for studies of KSHV lytic infection mechanisms. Key features • This protocol describes methods for culturing and antibiotically selecting iSLK-BAC16 cells for robust KSHV reactivation. • Use of flow cytometry to quantify KSHV reactivation rates. • Innovative use of automated plate readers to assess KSHV reactivation. Graphical overview <b>Schematic overview of the procedures used for general iSLK-BAC16 cell culture and Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation of iSLK-BAC16 cells.</b> A typical timeline of iSLK-BAC16 cell culture, antibiotic selection, KSHV reactivation, flow cytometry quantification, and plate reader assessment of KSHV lytic replication. Corresponding media requirements are denoted under the respective procedures. iSLK-BAC16 = doxycycline-inducible endothelial cells harboring the KSHV genome on an artificial bacterial chromosome; Dox = doxycycline; NaBu = sodium butyrate.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 11","pages":"e5334"},"PeriodicalIF":1.0,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12152104/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144287522","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 a Live Analysis System to Study Amyloplast Replication in <i>Arabidopsis</i> Ovule Integuments.","authors":"Makoto T Fujiwara, Rin Arakawa, Tomoko Abe, Ryuuichi D Itoh","doi":"10.21769/BioProtoc.5333","DOIUrl":"10.21769/BioProtoc.5333","url":null,"abstract":"<p><p>Amyloplasts, non-photosynthetic plastids specialized for starch synthesis and storage, proliferate in storage tissue cells of plants. To date, studies of amyloplast replication in roots and the ovule nucelli from various plant species have been performed using electron and fluorescence microscopy. However, a complete understanding of amyloplast replication remains unclear due to the absence of experimental systems capable of tracking their morphology and behavior in living cells. Recently, we demonstrated that <i>Arabidopsis</i> ovule integument could provide a platform for live-cell imaging of amyloplast replication. This system enables precise analysis of amyloplast number and shape, including the behavior of stroma-filled tubules (stromules), during proplastid-to-amyloplast development in post-mitotic cells. Here, we provide technical guidelines for observing and quantifying amyloplasts using conventional fluorescence microscopy in wild-type and several plastid-division mutants of <i>Arabidopsis</i>. Key features • Novel approach for investigating amyloplast differentiation and replication in plant cells. • Detection of stroma-labeled amyloplasts in whole-mount ovules using conventional fluorescence microscopy. • Facilitates quantitative and comparative analysis of amyloplast proliferation using various <i>Arabidopsis</i> resources. • Enables high-resolution analysis of changing amyloplast and stromule morphologies in living cells.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 11","pages":"e5333"},"PeriodicalIF":1.0,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12152115/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144287568","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":"Identification of S-locus F-box Protein Sequences in Diploid Potato, <i>Solanum okadae</i>, via Degenerate PCR.","authors":"Amar Hundare","doi":"10.21769/BioProtoc.5328","DOIUrl":"10.21769/BioProtoc.5328","url":null,"abstract":"<p><p>In many plant species, self-incompatibility (SI) is a mechanism that inhibits inbreeding. SI is gametophytic in the Solanaceae, with specificity determined by S-ribonucleases (S-RNases) in the pistil and S-locus F-box proteins (SLFs) in the pollen. The role of these proteins has been studied extensively in the Solanaceae, often using <i>Petunia</i> as a model. Using degenerate PCR and Sanger sequencing, this protocol identified three SLF sequences from self-incompatible diploid potato (<i>Solanum okadae</i>). While SLFs are well-characterized in model species like <i>Petunia</i>, there is limited sequence data and no standardized protocols for identifying SLFs in non-model species such as <i>S. okadae</i>, hindering broader insights into SI across the Solanaceae. This protocol fills that gap by using degenerate PCR and Sanger sequencing with primers designed from conserved <i>Petunia</i> SLF regions to identify SLF sequences in <i>S. okadae</i>. SLF sequences from 10 distinct Solanaceae members sharing maximum identity with the S2-haplotype of <i>Petunia</i> were used to design two pairs of primers targeting different regions of the target sequence. PCR amplification using designed degenerate primers yielded amplicons that were directly sequenced and joined together to get the partial SLF sequence. It was observed that the <i>S. okadae</i> shared an orthologous relation with the <i>Petunia</i> SLF according to the phylogenetic analysis. These SLFs could be used in future SI breakdown experiments via the competitive interaction route. This protocol, including the primer design, is novel for detecting SLF sequences in <i>S. okadae</i>. Key features • This protocol is applicable when the exact nucleotide sequence of the target DNA is not known but can be deduced from an amino acid sequence. • Straightforward, cost-effective, and can be used to find \"new\" genes or gene families. • Guidelines and a systematic approach for designing degenerate primers, along with a framework for annotating and comparing SLF genes within the Solanaceae family.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 11","pages":"e5328"},"PeriodicalIF":1.0,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12152105/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144287545","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":"Local Iontophoretic Application for Pharmacological Induction of Long-Term Synaptic Depression.","authors":"Borys Olifirov, Oleksandra Fedchenko, Alexandr Dovgan, Daria Babets, Volodymyr Krotov, Volodymyr Cherkas, Pavel Belan","doi":"10.21769/BioProtoc.5338","DOIUrl":"10.21769/BioProtoc.5338","url":null,"abstract":"<p><p>Long-term depression (LTD), a key form of synaptic plasticity, is typically induced through regulated Ca²⁺ entry via NMDA receptors and achieved by prolonged (up to hundreds of seconds) low-frequency presynaptic stimulation or bath application of NMDA receptor agonists. Electrophysiological approach to LTD induction requires specialized equipment, while bath applications limit productivity, as only one neuron per sample may be recorded. Here, we present a simple and effective protocol for pharmacological modeling of LTD in primary cultured neurons. This approach relies on highly localized iontophoretic application of NMDA, which induces LTD in individual cells, enhancing experimental throughput. We have analyzed spatio-temporal patterns of iontophoretic drug delivery and demonstrated how this technique may be combined with electrophysiological and live-cell imaging approaches to investigate LTD-related changes in synaptic strength and Ca²⁺-dependent signaling of neuronal Ca²⁺ sensor proteins. Key features • Easy, fast, and reliable induction of LTD in primary cultured neurons using iontophoretic NMDA application. • Suitable for the application of any ionic water-soluble compound and compatible with simultaneous multicolor fluorescence imaging and electrophysiological recording. • This protocol enables pharmacological targeting of individual neurons, substantially increasing experimental throughput.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 11","pages":"e5338"},"PeriodicalIF":1.0,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12152103/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144287548","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":"Mito Stress Assay of PBMCs With Seahorse XFe96 Flux Analyzer and Comparison of Poly-D-Lysine and Poly-L-Lysine for Cell Affinity.","authors":"Kumudu Subasinghe, Raymond Berry, Megan Rowe, Ali Winters, Shaohua Yang, Nicole Phillips","doi":"10.21769/BioProtoc.5327","DOIUrl":"10.21769/BioProtoc.5327","url":null,"abstract":"<p><p>The Seahorse 96 XF Analyzer (Agilent Technologies, Santa Clara, CA, USA) has been an effective tool in non-invasively measuring mitochondrial function for the past decade. It is a high-throughput respirometer that is considered the \"gold standard\" for quantifying mitochondrial function and bioenergetics in cells. Peripheral blood mononuclear cells (PBMCs) play a selective role in immune system responses and are key components of human immunity. Recent studies have suggested that these cell populations provide an overview of systemic changes within the body and therefore provide a source of sensitive biomarkers. Assessing mitochondrial function in PBMCs has been shown to provide an indication of metabolic stress associated with diseases such as diabetes and neurodegenerative conditions such as Alzheimer's disease. In this protocol, we use two adhesive compounds, Poly-D-Lysine (PDL) and Poly-L-Lysine (PLL), at 50 μg/mL each per well, to immobilize PBMCs to a specialized Seahorse microplate to perform mitochondrial stress assay using the Seahorse Analyzer. We compared six cell densities of PBMCs to identify the optimal cell density for use in Seahorse Mito Stress analysis. This protocol includes the immobilization of freshly isolated PBM cells into a Seahorse microplate, hydration and calibration of the sensor cartridge, cell seeding, running the Seahorse Analyzer for the Mito Stress test, and simple data analysis to compare the effectiveness of PLL and PDL as the coating agent for PBMCs. The data analysis indicates that there is no statistical difference between PLL and PDL. Key features • Designed for Seahorse 96-XF Analyzer, allowing it to work with lower cell densities and accommodate a greater number of replicates with high-throughput capabilities. • Two widely used cell adhesive compounds, Poly-D-Lysine and Poly-L-Lysine, are compared for their effectiveness in immobilizing PBMCs onto specialized Seahorse microplates. • The protocol takes two days to complete.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 11","pages":"e5327"},"PeriodicalIF":1.0,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12152111/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144287549","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}