Current protocols最新文献

{"title":"Streaming Long-Read Sequence Alignments for HLA Predictions Using HLAminer","authors":"René L. Warren,&nbsp;Inanc Birol","doi":"10.1002/cpz1.70124","DOIUrl":"https://doi.org/10.1002/cpz1.70124","url":null,"abstract":"<p>Long-read sequencing platforms such as the Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) platforms now offer sufficient read lengths, throughput, and accuracy at competitive costs to analyze polymorphic regions of the human genome, including the highly complex human leukocyte antigen (HLA) gene cluster—a cornerstone of human immunity. Here, we present a streamlined protocol for predicting HLA signatures from whole-genome shotgun (WGS) long-read sequencing data by directly streaming sequence alignments into HLAminer. This method is as simple as running minimap2, scales efficiently with the number of sequences, and works with any read aligner compatible with the SAM file format—eliminating the need to store bulky alignment files on disk. We provide a step-by-step guide for predicting HLA class I and class II alleles from third-generation long-read sequencing data and demonstrate the robustness of predictions even with older, less accurate WGS nanopore datasets and relatively low (10×) sequencing coverage. Code availability: HLAminer is available under the BC Cancer software license agreement (academic use) at https://github.com/bcgsc/HLAminer. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: HLA prediction from streamed ONT or PacBio long-read alignments</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70124","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143707570","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":"Isolation and Characterization of Human Tissue Resident Memory T cells","authors":"Isaac J. Jensen,&nbsp;Steven B. Wells,&nbsp;Julien Gras,&nbsp;Donna L. Farber","doi":"10.1002/cpz1.70120","DOIUrl":"https://doi.org/10.1002/cpz1.70120","url":null,"abstract":"<p>The majority of immune cells in the human body exist within the tissues rather than in the circulation. Nevertheless, most of our knowledge of the human immune system is biased towards the characterization and understanding of circulating immune cell populations because the latter are readily sampled, whereas cells in tissues are difficult to obtain and/or are limited to single sites of disease. Tissue-resident cells differ from circulating cells due to tissue-specific niche adaptations that influence their phenotype and function. For instance, T cells in tissues, resident memory (T_RM cells), exhibit tissue-specific properties that allow optimal protection from infection due to an acquired ability to coordinate rapid and efficacious pathogen clearance. Thus, to fully understand T-cell responses in various pathological conditions one must focus on the properties of T_RM cells and how they have been shaped by their environment. Moreover, one must sample and analyze T cells from multiple tissues, optimally from the same individual, to determine how infectious, cancer, or autoimmune challenge is affecting homeostatic function. Our longstanding collaboration with the organ procurement organization, LiveOnNY, provides unique access to multiple lymphoid, mucosal, and peripheral tissues from organ donors where consent for research use has been obtained. These samples have enabled characterization of human tissue resident memory T cells and other immune cell types across a variety of tissues. Concomitant with this endeavor, we developed and refined a series of methodologies critical for extracting immune cells from tissue for the purpose of phenotypic and mechanistic interrogation. Here, we describe our optimized protocols for processing select human tissues and the requisite coordination and considerations for their maximal yield and tissue quality. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Isolation of immune cells from blood-rich sites [spleen (SPL), blood (BLD), bone marrow (BOM)]</p><p><b>Basic Protocol 2</b>: Isolation of immune cells from lymph nodes, tonsils, and thymus [iliac lymph nodes (ILN), lung lymph nodes (LLN), mesenteric lymph nodes (MLN), colonic lymph nodes (CLN), hepatic lymph nodes (HLN), tonsils (TON), thymus (THY)]</p><p><b>Basic Protocol 3</b>: Isolation of immune cells from the lungs [lung (LNG), bronchioalveolar lavage (BAL)]</p><p><b>Basic Protocol 4</b>: Isolation of immune cells from the intestines [jejunum epithelial layer (JEL), jejunum lamina propria (JLP), colon epithelial layer (CEL), colon lamina propria (CLP)]</p><p><b>Basic Protocol 5</b>: Isolation of immune cells from the liver (LVR)</p><p><b>Basic Protocol 6</b>: Immune cell staining for flow cytometry</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143707569","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction: Establishment and Culture of Human Intestinal Organoids Derived from Adult Stem Cells","authors":"Cayetano Pleguezuelos-Manzano,&nbsp;Jens Puschhof,&nbsp;Stieneke van den Brink,&nbsp;Veerle Geurts,&nbsp;Joep Beumer,&nbsp;Hans Clevers","doi":"10.1002/cpz1.70132","DOIUrl":"https://doi.org/10.1002/cpz1.70132","url":null,"abstract":"<p><i>Current Protocols</i> is issuing corrections for the following protocol article.</p><p>Pleguezuelos-Manzano, C., Puschhof, J., van den Brink, S., Geurts, V., Beumer, J., &amp; Clevers, H. (2020). Establishment and culture of human intestinal organoids derived from adult stem cells. <i>Current Protocols in Immunology</i>, <i>130</i>, e106. doi: 10.1002/cpim.106</p><p>In the above-referenced article:</p><p>In step 3 of Basic Protocol 1, collagenase concentration has been changed from “5 mg/ml” to “50 µg/ml”.</p><p>The current version online now includes this correction and may be considered the authoritative version of record.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70132","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143707480","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":"Extracting Protoplasts from Filamentous Fungi Using Extralyse, An Enzyme Used in the Wine Industry","authors":"Andi Wilson,&nbsp;Alida van Dijk,&nbsp;Bianke Marx,&nbsp;Deanne du Plessis,&nbsp;Grant Terblanche,&nbsp;Simoné Bornman,&nbsp;P. Markus Wilken,&nbsp;Tuan A. Duong,&nbsp;Henrik H. De Fine Licht,&nbsp;Brenda D. Wingfield","doi":"10.1002/cpz1.70122","DOIUrl":"https://doi.org/10.1002/cpz1.70122","url":null,"abstract":"<p>The ability to extract protoplasts has contributed significantly to the study of fungi and plants. Protoplasts have historically been used to determine chromosome number via pulsed-field electrophoresis and for the functional characterization of genes via protoplast transformation. More recently, protoplasts have been used to extract the high-molecular-weight DNA required for long-read sequencing projects. The availability of efficient protoplast extraction protocols is thus integral to the study and experimental manipulation of model and non-model fungi. One major hurdle to the development of such protocols has been the discontinuation of enzymes and enzyme cocktails used to digest the fungal cell wall. Here, we provide five protoplast extraction protocols for use in various filamentous ascomycete species spanning the genera <i>Ceratocystis</i>, <i>Fusarium</i>, <i>Metarhizium</i>, <i>Ophiostoma</i>, and <i>Sclerotinia</i>. These protocols all use an inexpensive, readily available enzyme cocktail called Extralyse, a commercially available product commonly used in the wine making industry. Using this enzyme cocktail overcomes reliance on the laboratory-grade enzymes that have frequently been discontinued and are often cost prohibitive at the concentrations required. The protocols described here will allow further research, including genome editing, to be conducted in these fungal genera. Importantly, these protocols also provide a starting point for the development of protoplast extraction techniques in other filamentous fungi. This resource can therefore be used to expand the molecular toolkits available for fungi beyond the species described here, including those with relevance in both medical and biotechnological industries. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Protoplast extractions from <i>Ceratocystis eucalypticola</i> and <i>Ceratocystis fimbriata</i></p><p><b>Basic Protocol 2</b>: Protoplast extractions from <i>Fusarium circinatum</i></p><p><b>Basic Protocol 3</b>: Protoplast extractions from <i>Metarhizium acridum</i>, <i>Metarhizium brunneum</i>, and <i>Metarhizium guizhouense</i></p><p><b>Basic Protocol 4</b>: Protoplast extractions from <i>Ophiostoma novo-ulmi</i></p><p><b>Basic Protocol 5</b>: Protoplast extractions from <i>Sclerotinia sclerotiorum</i></p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70122","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143689810","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":"Verifying Proper Function of the Aerosol Evacuation System Prior to Sorting Potentially Infectious Samples","authors":"Kristen M. Reifel,&nbsp;Avrill Aspland,&nbsp;Suat Dervish,&nbsp;Iyadh Douagi,&nbsp;Alyssa C. Fears,&nbsp;Evan R. Jellison,&nbsp;Cecily C. Midkiff,&nbsp;Taryn Mockus-Daehn,&nbsp;Matilda J. Moström,&nbsp;Michael Solga,&nbsp;Brandon K. Swan,&nbsp;James Thomas,&nbsp;Stephen Perfetto","doi":"10.1002/cpz1.70123","DOIUrl":"10.1002/cpz1.70123","url":null,"abstract":"<p>High concentrations of aerosols can be generated within the sort collection area of cell sorters during instrument failures that cause the stream to deviate, such as a partial nozzle obstruction. Complete containment of these aerosol particles becomes essential for operator safety when working with potentially infectious or hazardous samples. Currently, aerosol containment is accomplished through the generation of continuous negative airflow within the sort collection area using an aerosol evacuation system, which can be enhanced by using primary containment devices such as biosafety cabinets. Unlike biosafety cabinets, many aerosol evacuation systems are not certified or tested on a regular basis after installation. Therefore, proper function of the system must be verified by the user prior to running hazardous samples to ensure that it is operational and provides sufficient protection for the operator. This protocol describes an updated procedure for verifying the containment and evacuation of aerosols generated when the stream is disrupted during an instrument failure. In this procedure, aerosols are generated to simulate a partial nozzle obstruction while running 1-µm fluorescent beads. Air samples are collected just outside the sort collection area using a disposable impactor-style aerosol sampler cassette and are examined for the presence of beads in an effort to detect aerosols. If no beads are present, aerosols were adequately contained and evacuated by the aerosol evacuation system. The presence of beads, however, indicates a potential failure of the aerosol evacuation system and/or other engineering controls that could result in the exposure of laboratory workers to any infectious or hazardous samples that are run through the instrument. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.</p><p><b>Basic Protocol</b>: Validation of the aerosol evacuation system using 1-µm fluorescent beads and disposable aerosol sampler cassettes</p><p><b>Support Protocol</b>: Preparation of 1-µm fluorescent bead reference slide</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11927378/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143675099","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":"Protocol for Controlling the Strand Selectivity of siRNA Using Acyclic Artificial Nucleic Acids","authors":"Jumpei Ariyoshi,&nbsp;Hiroyuki Asanuma,&nbsp;Yukiko Kamiya","doi":"10.1002/cpz1.70103","DOIUrl":"10.1002/cpz1.70103","url":null,"abstract":"<p>Small interfering RNA (siRNA) has emerged as a promising therapeutic candidate against previously intractable diseases. An effective siRNA must have high on-target activity while off-target effects are minimized. This balance can be achieved by enhancing the selectivity of the antisense strand through sequence optimization and appropriate chemical modifications. Acyclic artificial nucleic acids such as serinol nucleic acids (SNA) have demonstrated on-target activity while suppressing off-target effects. This article provides guidelines for designing SNA-modified siRNA and outlines a method for the experimental evaluation of the on-target and off-target activities of siRNAs, ensuring accurate functional validation in cell systems. These protocols benefit researchers developing siRNA-based therapeutics to optimize siRNA selectivity and efficacy while minimizing off-target effects through innovative design strategies. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Design of SNA-modified siRNA</p><p><b>Basic Protocol 2</b>: Design and preparation of vector plasmids using inverse PCR</p><p><b>Alternate Protocol</b>: Design and preparation of vector plasmid using restriction enzymes and ligase</p><p><b>Basic Protocol 3</b>: Evaluation of the on- and off-target effects of siRNAs using the dual-luciferase assay</p><p><b>Support Protocol 1</b>: Agarose gel electrophoresis and protocol for purifying DNA from gels</p><p><b>Support Protocol 2</b>: Transformation and amplification of plasmids</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143664892","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthesis of Oligodeoxynucleotide Containing Pseudo-Deoxycytidine and Its Triphosphate Derivative","authors":"Ryo Miyahara,&nbsp;Yosuke Taniguchi","doi":"10.1002/cpz1.70101","DOIUrl":"https://doi.org/10.1002/cpz1.70101","url":null,"abstract":"<p>This article describes a detailed synthetic protocol for the preparation of oligodeoxynucleotide (ODN) containing pseudo-deoxycytidine (ψdC) and its triphosphate derivative (ψdCTP). These molecules were synthesized as novel compounds that recognize iso-2'-deoxyguanosine (iso-dG) in DNA. Iso-dG is one of the tautomers of 2-hydroxy-2'-deoxyadenosine (2-OH-dA), which is known as an oxidatively damaged nucleobase, and its selective recognition in DNA is expected to play a very important role in the diagnosis and pathogenesis of diseases. The hydroxyl groups of the known glycal compound were protected with silyl groups, and then coupled with 5-iodouracil under Mizorogi-Heck reaction conditions, yielding ψdU after desilylation and diastereoselective reduction. The endocyclic amino group of ψdU was protected by the benzyl group. Subsequently, the carbonyl group at the 6-position of the nucleobase was activated and converted to an amino group through treatment with aqueous ammonia. The benzyl group was removed, and the exocyclic amino group was protected with a benzoyl group. On one hand, the silyl groups at the 3’ and 5’ positions were deprotected, converted into a phosphoramidite unit, and incorporated into an ODN. On the other hand, the hydroxyl group at the 5’ position was selectively deprotected and then directly converted into the triphosphate using Van Boom's reagent under acidic conditions. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Synthesis of ODNs having ψdC and ψdCTP</p><p><b>Basic Protocol 2</b>: Melting temperature of duplex formation between ODNs containing ψdC unit and 2-OH-dA</p><p><b>Basic Protocol 3</b>: A single nucleotide primer extension reaction of ψdCTP for a template strand containing 2-OH-dA</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70101","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143632700","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":"Expression and Purification of SARS-related Spike Glycoproteins for Cryo-EM Analysis","authors":"Francesca R. Hills,&nbsp;Fátima Jorge,&nbsp;Laura N. Burga,&nbsp;Mihnea Bostina","doi":"10.1002/cpz1.70115","DOIUrl":"https://doi.org/10.1002/cpz1.70115","url":null,"abstract":"<p><i>Coronaviridae</i> spike glycoproteins mediate viral entry and fusion to host cells through binding to host receptors (i.e., ACE2, DPP4) and are key components in determining viral host range, making them targets for antiviral research. Here, we describe the expression, purification, and characterization of recombinant spike proteins to aid in protein characterization and analysis. These protocols were used for the production of spike glycoproteins from civet, pangolin, and bat coronaviruses, as well as high-resolution cryo-electron microscopy (cryo-EM) structural analysis of bat and civet host coronavirus spike glycoproteins (Hills et al., 2024). © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Expression and purification of SARS-CoV spike protein from ExpiCHO cells</p><p><b>Basic Protocol 2</b>: Preparation of SARS-CoV spike protein for visualization by negative-stain transmission electron microscopy and cryo-electron microscopy</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70115","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143571348","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 Physiologically Relevant In Vitro Model of Nonreplicating Persistent Mycobacterium tuberculosis in Caseum","authors":"Min Xie,&nbsp;Paulina Osiecki,&nbsp;Suyapa Rodriguez,&nbsp;Véronique Dartois,&nbsp;Jansy Sarathy","doi":"10.1002/cpz1.70118","DOIUrl":"https://doi.org/10.1002/cpz1.70118","url":null,"abstract":"<p>Tuberculosis (TB) remains one of the leading infectious causes of death worldwide. Persistent bacterial populations in specific microenvironments within the host hamper efficient TB chemotherapy. Caseum in the necrotic core of closed granulomas and cavities of pulmonary TB patients can harbor high burdens of drug-tolerant <i>Mycobacterium tuberculosis</i> (MTB) bacilli, making them particularly difficult to sterilize. Here, we describe protocols for the generation of a surrogate matrix using lipid-rich macrophages to mimic the unique composition of caseum <i>in vivo</i>. Importantly, this caseum surrogate induces metabolic and physiological changes within MTB that reproduce the nonreplicating drug-tolerant phenotype of the pathogen in the native caseous environment, making it advantageous over alternative <i>in vitro</i> models of nonreplicating persistent (NRP) MTB. The protocols include culture of THP-1 monocytes, stimulation of lipid droplet accumulation, lysis and denaturation of the foamy macrophages, inoculation and preadaptation of MTB bacilli in the caseum surrogate, and evaluation of drug bactericidal activity against the NRP population. This novel <i>in vitro</i> model is being used to screen for potent bactericidal antimicrobial agents and to identify vulnerable drug targets, among a variety of other applications, thereby reducing our reliance on <i>in vivo</i> models. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Caseum surrogate preparation from γ-irradiated <i>M. tuberculosis</i>–induced foamy THP-1 monocyte–derived macrophages (THPMs)</p><p><b>Alternate Protocol 1</b>: Caseum surrogate preparation from stearic acid–induced THPMs</p><p><b>Basic Protocol 2</b>: Generation of nonreplicating persistent <i>M. tuberculosis</i> and drug susceptibility testing</p><p><b>Alternate Protocol 2</b>: Higher-throughput drug susceptibility screening using caseum surrogate</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70118","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143571349","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":"Optimized, Efficient Measurement of the Expression of Undifferentiated Stem Cell Markers in Human Induced Pluripotent Stem Cells (iPSCs) by Flow Cytometry","authors":"Vaishanavi Saware,&nbsp;Wendy Runyon,&nbsp;Sam Hu,&nbsp;Benjamin van Soldt,&nbsp;Ritu Kumar,&nbsp;Jane Srivastava","doi":"10.1002/cpz1.70105","DOIUrl":"https://doi.org/10.1002/cpz1.70105","url":null,"abstract":"<p>Induced pluripotent stem cells (iPSCs) have revolutionized the fields of regenerative medicine, disease modeling, and drug discovery. However, the usage of iPSCs for various applications has been hampered by the observed line-to-line variability in their differentiation capacity. Therefore, it is important to verify the pluripotent status of iPSCs. A very effective way to define the pluripotent state of iPSCs is by evaluating the expression of established undifferentiated stem cell markers. A bona fide iPSC must have high, homogeneous expression of these markers. Here, we present a cost-effective platform that can be readily utilized by researchers to define the pluripotency status of iPSCs by measuring the expression of surface and intracellular markers by flow cytometry. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: iPSC culture and collection for flow cytometry analysis</p><p><b>Basic Protocol 2</b>: Staining of iPSCs for extracellular and intracellular undifferentiated stem cell markers</p><p><b>Basic Protocol 3</b>: Flow cytometry acquisition</p><p><b>Basic Protocol 4</b>: Flow cytometry data analysis</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143530157","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}