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Protocol for fabricating elastomeric stencils for patterned stem cell differentiation. 用于干细胞模式化分化的弹性模板制造规程。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-11-26 DOI: 10.1016/j.xpro.2024.103187
Stefanie Lehr, Jack Merrin, Monika Kulig, Thomas George Minchington, Anna Kicheva
{"title":"Protocol for fabricating elastomeric stencils for patterned stem cell differentiation.","authors":"Stefanie Lehr, Jack Merrin, Monika Kulig, Thomas George Minchington, Anna Kicheva","doi":"10.1016/j.xpro.2024.103187","DOIUrl":"10.1016/j.xpro.2024.103187","url":null,"abstract":"<p><p>Geometrically controlled stem cell differentiation promotes reproducible pattern formation. Here, we present a protocol to fabricate elastomeric stencils for patterned stem cell differentiation. We describe procedures for using photolithography to produce molds, followed by molding polydimethylsiloxane (PDMS) to obtain stencils with through holes. We then provide instructions for culturing cells on stencils and, finally, removing stencils to allow colony growth and cell migration. This approach yields reproducible two-dimensional organoids tailored for quantitative studies of growth and pattern formation. For complete details on the use and execution of this protocol, please refer to Lehr et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103187"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11626805/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142740560","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
Protocol for simultaneous detection of mRNAs and (phospho-)proteins with ARTseq-FISH in mouse embryonic stem cells. 利用 ARTseq-FISH 同时检测小鼠胚胎干细胞中 mRNA 和（磷酸）蛋白的方法。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-01 DOI: 10.1016/j.xpro.2024.103336
Xinyu Hu, Bob van Sluijs, Óscar García-Blay, Wilhelm T S Huck, Maike M K Hansen
{"title":"Protocol for simultaneous detection of mRNAs and (phospho-)proteins with ARTseq-FISH in mouse embryonic stem cells.","authors":"Xinyu Hu, Bob van Sluijs, Óscar García-Blay, Wilhelm T S Huck, Maike M K Hansen","doi":"10.1016/j.xpro.2024.103336","DOIUrl":"10.1016/j.xpro.2024.103336","url":null,"abstract":"<p><p>Understanding the molecular signatures of individual cells within complex biological systems is crucial for deciphering cellular heterogeneity and uncovering regulatory mechanisms. Here, we present a protocol for simultaneous multiplexed detection of selected mRNAs and (phospho-)proteins in mouse embryonic stem cells using spatial single-cell profiling. We describe steps for employing single-stranded DNA (ssDNA)-labeled antibo'dies, padlock probes, and rolling circle amplification to achieve simultaneous visualization of mRNAs and (phospho-)proteins at subcellular resolution. This protocol has potential application in identifying cells in heterogeneous biological microenvironments. For complete details on the use and execution of this protocol, please refer to Hu et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103336"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11472615/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142366779","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
Protocol for CRISPR-Cas9 genome editing of a swine cell line via electroporation. 通过电穿孔技术对猪细胞系进行 CRISPR-Cas9 基因组编辑的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-10 DOI: 10.1016/j.xpro.2024.103385
Patricia Kiesler, Stella S Lee, Alexis L Norris, Mayumi F Miller, Carlo J Mercado, Adam L Moyer, Samantha Maragh
{"title":"Protocol for CRISPR-Cas9 genome editing of a swine cell line via electroporation.","authors":"Patricia Kiesler, Stella S Lee, Alexis L Norris, Mayumi F Miller, Carlo J Mercado, Adam L Moyer, Samantha Maragh","doi":"10.1016/j.xpro.2024.103385","DOIUrl":"10.1016/j.xpro.2024.103385","url":null,"abstract":"<p><p>Genome editing technology is being used in animals for a variety of purposes, including improvement of animal and public health outcomes. Characterization of genome editing reagents and anticipated genomic alterations is an essential step toward the development of an edited animal. Here, we present a protocol for genome editing in the swine testicular (ST) cell line. We describe steps for evaluating CRISPR-Cas9 complex functionality in vitro, delivering editing molecules into cells by transfection, and assessing target editing via Sanger sequencing.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103385"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142406972","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}
引用次数: 0
Protocol for generating high-fidelity proteomic profiles using DROPPS. 使用 DROPPS 生成高保真蛋白质组图谱的程序。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-18 DOI: 10.1016/j.xpro.2024.103397
Matthew Waas, Meinusha Govindarajan, Amanda Khoo, Charlotte Zuo, Aastha Aastha, Jilin He, Michael Woolman, Annie Ha, Brian Lin, Thomas Kislinger
{"title":"Protocol for generating high-fidelity proteomic profiles using DROPPS.","authors":"Matthew Waas, Meinusha Govindarajan, Amanda Khoo, Charlotte Zuo, Aastha Aastha, Jilin He, Michael Woolman, Annie Ha, Brian Lin, Thomas Kislinger","doi":"10.1016/j.xpro.2024.103397","DOIUrl":"10.1016/j.xpro.2024.103397","url":null,"abstract":"<p><p>Deep mass spectrometry-based proteomic profiling of rare cell populations has been constrained by sample input requirements. Here, we present a protocol for droplet-based one-pot preparation for proteomic samples (DROPPS), an accessible low-input platform that generates high-fidelity proteomic profiles of 100-2,500 cells. We describe steps for depositing cellular material, cell lysis, and digesting proteins in the same microliter-droplet well. We anticipate DROPPS will accelerate biology-driven proteomic research for a multitude of rare cell populations. For complete details on the use and execution of this protocol, please refer to Waas et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103397"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11513556/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142476477","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
Protocol for intracellular immunofluorescence measurements of latent HIV reactivation in a primary CD4+ T cell model. 初级 CD4+ T 细胞模型中潜伏 HIV 再激活的细胞内免疫荧光测量规程。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-18 DOI: 10.1016/j.xpro.2024.103398
Uri Mbonye, Anna Agaponova, Muda Yang, Jonathan Karn
{"title":"Protocol for intracellular immunofluorescence measurements of latent HIV reactivation in a primary CD4<sup>+</sup> T cell model.","authors":"Uri Mbonye, Anna Agaponova, Muda Yang, Jonathan Karn","doi":"10.1016/j.xpro.2024.103398","DOIUrl":"10.1016/j.xpro.2024.103398","url":null,"abstract":"<p><p>Investigating the molecular mechanisms of HIV latency reversal in a proper physiological context can only be done in primary cells. Here, we describe a primary T cell model of HIV latency and a reliable flow cytometry assay to measure latency reversal efficacy by dual immunofluorescence staining for Nef and Tat. We also describe a procedure for identifying latency-reversing agents that effectively induce the biogenesis of P-TEFb, an obligate host transcription factor for HIV, while monitoring their effects on T cell activation.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103398"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11513541/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142476479","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
Protocol to identify E3 ligases amenable to biodegraders using a cell-based screening. 利用基于细胞的筛选来确定适合生物降解的 E3 连接酶的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-24 DOI: 10.1016/j.xpro.2024.103413
Marie Sorbara, Margot Cristol, Anaïs Cornebois, Klervi Desrumeaux, Pierre Cordelier, Nicolas Bery
{"title":"Protocol to identify E3 ligases amenable to biodegraders using a cell-based screening.","authors":"Marie Sorbara, Margot Cristol, Anaïs Cornebois, Klervi Desrumeaux, Pierre Cordelier, Nicolas Bery","doi":"10.1016/j.xpro.2024.103413","DOIUrl":"10.1016/j.xpro.2024.103413","url":null,"abstract":"<p><p>Here, we provide a protocol for the identification of E3 ubiquitin ligases that are functional when implemented as biodegraders using a cell-based screening assay. We describe steps for establishing a stable cell line expressing a GFP-tagged protein of interest (POI), preparing a sub-library of E3 ligases to screen, and performing the cell-based screening. This protocol can be broadly applied to identify any functional E3 ligase in a biodegrader setting. For complete details on the use and execution of this protocol, please refer to Cornebois et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103413"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11541768/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142509489","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
Protocol for Cas9-targeted long-read sequencing in Globodera pallida and Globodera rostochiensis. Globodera pallida 和 Globodera rostochiensis 的 Cas9 靶向长序列测序规程。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-11-01 DOI: 10.1016/j.xpro.2024.103427
Unnati Sonawala, Lida Derevnina, Sebastian Eves-van den Akker
{"title":"Protocol for Cas9-targeted long-read sequencing in Globodera pallida and Globodera rostochiensis.","authors":"Unnati Sonawala, Lida Derevnina, Sebastian Eves-van den Akker","doi":"10.1016/j.xpro.2024.103427","DOIUrl":"10.1016/j.xpro.2024.103427","url":null,"abstract":"<p><p>We present a protocol to achieve a higher depth of long-read sequencing of region(s) of interest in potato cyst nematodes without amplification using a Cas9-based Nanopore enrichment approach. We describe steps for designing high-fidelity guide RNAs to be used with Cas9 nuclease, extracting high-molecular-weight DNA from the nematodes, and dephosphorylating genomic DNA ends. We then detail procedures for using Cas9-guide RNA complex to make targeted cleavage of the region of interest followed by a Nanopore library preparation. For complete details on the use and execution of this protocol, please refer to Sonawala et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103427"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11566856/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142565110","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
Protocol for manufacturing photosynthetic cyanobacterial cell-based living building materials. 制造基于光合蓝藻细胞的活体建筑材料的规程。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-11-02 DOI: 10.1016/j.xpro.2024.103426
Yongjun Son, Jihyeon Min, Jiyoon Park, Chongku Yi, Woojun Park
{"title":"Protocol for manufacturing photosynthetic cyanobacterial cell-based living building materials.","authors":"Yongjun Son, Jihyeon Min, Jiyoon Park, Chongku Yi, Woojun Park","doi":"10.1016/j.xpro.2024.103426","DOIUrl":"10.1016/j.xpro.2024.103426","url":null,"abstract":"<p><p>Cement-free and photosynthetic cyanobacterial cell-based living building materials (LBMs) can be manufactured using microbially induced calcium carbonate precipitation (MICP) technology. Here, we present a protocol for cultivating Leptolyngbya boryana GGD and manufacturing LBMs using a sand-gelatin solution. We describe steps for fabricating acrylic molds and mixing abiotic substances with cyanobacterial cells for constructing LBMs. We then detail the procedures for analyzing the compressive strengths of our LBMs with or without the cyanobacterial cells using a universal testing machine (UTM). For complete details on the use and execution of this protocol, please refer to Son et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103426"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11567024/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142568171","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
Protocol for studying macrophage lipid crosstalk with murine tumor cells. 研究巨噬细胞与小鼠肿瘤细胞脂质串扰的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-11-02 DOI: 10.1016/j.xpro.2024.103421
Daan J Kloosterman, Martina Farber, Menno Boon, Johanna Erbani, Leila Akkari
{"title":"Protocol for studying macrophage lipid crosstalk with murine tumor cells.","authors":"Daan J Kloosterman, Martina Farber, Menno Boon, Johanna Erbani, Leila Akkari","doi":"10.1016/j.xpro.2024.103421","DOIUrl":"10.1016/j.xpro.2024.103421","url":null,"abstract":"<p><p>Lipid accumulation has recently emerged as a key feature underlying the pro-tumorigenic role of macrophages. Here, we present a workflow to study macrophage lipid crosstalk with tumor cells. We describe steps for the identification, purification, and multi-omics characterization of lipid-laden macrophages (LLMs) from murine tumors and outline protocols to assess the functional significance of LLMs in cancer malignancy. This approach has the potential to uncover the source of lipids that drives LLM formation and its pro-tumorigenic potential in multiple cancer types. For complete details on the use and execution of this protocol, please refer to Kloosterman, Erbani, et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103421"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11566343/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142568570","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
Protocol for transformation-free genome editing in plants using RNA virus vectors for CRISPR-Cas delivery. 利用 RNA 病毒载体传递 CRISPR-Cas 进行植物免转化基因组编辑的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-11-05 DOI: 10.1016/j.xpro.2024.103437
Huanhuan Lou, Haiying Xiang, Wanli Zeng, Jiarui Jiang, Jianduo Zhang, Li Xu, Chenglu Zhao, Qian Gao, Zhenghe Li
{"title":"Protocol for transformation-free genome editing in plants using RNA virus vectors for CRISPR-Cas delivery.","authors":"Huanhuan Lou, Haiying Xiang, Wanli Zeng, Jiarui Jiang, Jianduo Zhang, Li Xu, Chenglu Zhao, Qian Gao, Zhenghe Li","doi":"10.1016/j.xpro.2024.103437","DOIUrl":"10.1016/j.xpro.2024.103437","url":null,"abstract":"<p><p>Plant virus vectors have emerged as promising tools for CRISPR-Cas reagent delivery. Here, we present a protocol for DNA-free plant genome editing using an engineered RNA virus vector for the transient delivery of CRISPR-Cas components. We describe steps for viral vector construction, viral vector recovery through agroinoculation of Nicotiana benthamiana, mechanical inoculation of target plant hosts, analysis of somatic mutagenesis frequency, and regeneration of mutant plants. The method achieves high editing efficiency and eliminates the need for stable plant transformation. For complete details on the use and execution of this protocol, please refer to Liu et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103437"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11577223/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142591536","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
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