Methods in cell biologyPub Date : 2024-01-01Epub Date: 2022-10-31DOI: 10.1016/bs.mcb.2022.09.004
Suzita Mohd Noor, Chee Ern David Wong, Pooi-Fong Wong, Anwar Norazit
{"title":"Generation of glial cell-derived neurotrophic factor (gdnf) morphants in zebrafish larvae by cerebroventricular microinjection of vivo morpholino.","authors":"Suzita Mohd Noor, Chee Ern David Wong, Pooi-Fong Wong, Anwar Norazit","doi":"10.1016/bs.mcb.2022.09.004","DOIUrl":"10.1016/bs.mcb.2022.09.004","url":null,"abstract":"<p><p>Dopaminergic neurons in the brain are an important source of dopamine, which is a crucial neurotransmitter for wellbeing, memory, reward, and motor control. Deficiency of dopamine due to advanced age and accumulative dopaminergic neuron defects can lead to movement disorders such as Parkinson's disease. Glial cell-derived neurotrophic factor (GDNF) is one of many factors involved in dopaminergic neuron development and/or survival. However, other endogenous GDNF functions in the brain await further investigation. Zebrafish is a well-established genetic model for neurodevelopment and neurodegeneration studies. Importantly, zebrafish shares approximately 70% functional orthologs with human genes including GDNF. To gain a better understanding on the precise functional role of gdnf in dopaminergic neurons, our laboratory devised a targeted knockdown of gdnf in the zebrafish larval brain using vivo morpholino. Here, detailed protocols on the generation of gdnf morphants using vivo morpholino are outlined. This method can be applied for targeting of genes in the brain to determine specific spatiotemporal gene function in situ.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139672172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Methods for detection of mitochondrial reactive oxygen species in senescent cells.","authors":"Fenniche Salma, Oubaddou Yassire, Bakri Youssef, Dupuy Corinne, Rabii Ameziane El Hassani","doi":"10.1016/bs.mcb.2022.09.011","DOIUrl":"10.1016/bs.mcb.2022.09.011","url":null,"abstract":"<p><p>Cellular senescence is a pathophysiological process with multifaceted effects. It is involved in wound healing, aging and age-related diseases as well as cancer. On the one hand, senescence is considered as barrier against tumorigenesis by inducing an irreversible/prolonged cell cycle arrest. On the other hand, it may promote tumorigenesis when senescent cells accumulate genomic instability and bypass this cell cycle arrest. Interestingly, the bystander effects mediate the propagation of the genetic instability from senescent cells to their environment through the SASP (Senescence Associated Secretory Phenotype) including proinflammatory cytokines, proteases, growth factors and Reactive Oxygen Species 'ROS.' From several markers explored to detect senescent cells (β-galactosidase, p16, p21, p53, heterochromatin foci, DNA damage,…), ROS arouse particular interest because of their involvement at the chronic supraphysiological level, in the induction and maintain of DNA damage, inflammation, cell cycle disruption and epigenetic instability. In this context, the choice of methods to detect ROS in senescent cells is of particular interest and must take into account relevant parameters as well as the specificity for each species of ROS and the subcellular localization of ROS production. In this chapter, we introduce senescence and ROS, we briefly discuss the advantages and the shortcomings of methods routinely used to detect ROS. In addition, we describe the protocol to detect ROS at mitochondrial level (using the MitoSOX staining) in the BCPAP cell line (from human papillary thyroid carcinomas) expressing BRAF<sup>V600E</sup> oncogene known to trigger senescence.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139672196","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Methods in cell biologyPub Date : 2024-01-01Epub Date: 2023-03-25DOI: 10.1016/bs.mcb.2023.02.005
Natasha Ramakrishnan, Taylor Malachowski, Priyanka Verma
{"title":"A high-content flow cytometry and dual CRISPR-Cas9 based platform to quantify genetic interactions.","authors":"Natasha Ramakrishnan, Taylor Malachowski, Priyanka Verma","doi":"10.1016/bs.mcb.2023.02.005","DOIUrl":"10.1016/bs.mcb.2023.02.005","url":null,"abstract":"<p><p>Probing epistasis between two genes can be a critical first step in identifying the molecular players in a cellular pathway. The advent of CRISPR-Cas mediated genetic screen has enabled studying of these genetic interactions at a genomic scale. However, when combining depletion of two genes using CRISPR Cas9, reduced targeting efficiencies due to competition for Cas loading and recombination in the cloning step have emerged as key challenges. Moreover, given conventional CRISPR screens typically involve comparison between the initial and final time point, it is difficult to parse the time kinetics with which a perturbed genetic interaction impacts viability, and it also becomes challenging to assess epistasis with essential genes. Here, we discuss a high-throughput flow-based approach to study genetic interactions. By utilizing two different Cas9 orthologs and monitoring viability at multiple time points, this approach helps to effectively mitigate the limitations of Cas9 competition and enables assessment of genetic interactions with both essential and non-essential genes at a high temporal resolution.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139741336","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Methods in cell biologyPub Date : 2024-01-01Epub Date: 2023-07-03DOI: 10.1016/bs.mcb.2023.05.012
Maxime Lalonde, Henning Ummethum, Manuel Trauner, Andreas Ettinger, Stephan Hamperl
{"title":"An automated image analysis pipeline to quantify the coordination and overlap of transcription and replication activity in mammalian genomes.","authors":"Maxime Lalonde, Henning Ummethum, Manuel Trauner, Andreas Ettinger, Stephan Hamperl","doi":"10.1016/bs.mcb.2023.05.012","DOIUrl":"10.1016/bs.mcb.2023.05.012","url":null,"abstract":"<p><p>Transcription-replication conflicts (TRCs) represent a potent endogenous source of replication stress. Besides the spatial and temporal coordination of replication and transcription programs, cells employ many additional mechanisms to resolve TRCs in a timely manner, thereby avoiding replication fork stalling and genomic instability. Proximity ligation assays (PLA) using antibodies against actively elongating RNA Polymerase II (RNAPIIpS2) and PCNA to detect proximity (<40nm) between transcribing RNA polymerases and replication forks can be used to assess and quantify TRC levels in cells. A complementary fluorescence microscopy approach to assess the spatial coordination of transcription and replication activities in the nucleus is to quantify the colocalization (200-400nm) between active transcription and ongoing replication using immunofluorescence staining with an antibody against elongating RNA Polymerase II (RNAPIIpS2) and EdU-Click-it pulse-labelling, respectively. Despite significant efforts to automate image analysis, the need for manual verification, correction, and complementation of automated processes creates a bottleneck for efficient, high-throughput and large-scale imaging. Here, we describe an automated Fiji image analysis macro that allows the user to automate the measurement of RNAPIIpS2 and EdU levels and extract the key parameters such as transcription-replication (TR) colocalization and TRC-PLA foci count from single cells in a high throughput manner. While we showcase the usability of this analysis pipeline for quantifying TR colocalization and TRC-PLA in mouse embryonic stem cells (mESCs), the analysis pipeline is designed as a generally applicable tool allowing the quantification of nuclear signals, colocalization and foci count in various model systems and cell types.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139741337","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Enrichment of DNA replication intermediates by EdU pull down.","authors":"Fabio Pessina, Alessia Romussi, Daniele Piccini, Giulia Mazzucco, Mario Varasi, Ylli Doksani","doi":"10.1016/bs.mcb.2022.11.001","DOIUrl":"10.1016/bs.mcb.2022.11.001","url":null,"abstract":"<p><p>Analysis of replication fork structures in electron microscopy (EM) can provide important mechanistic insights in DNA replication studies. A major challenge in this type of analysis is the paucity of replication intermediates. At any given time only a small fraction of the restriction fragments of genomic DNA will contain a replication fork. To address this issue, we have developed an EdU-pull-down procedure to enrich for replicating DNA. Cells are exposed to a brief pulse of EdU, a cleavable biotin moiety is attached to EdU by copper-catalyzed azide-alkyne cycloaddition (CuAAC), in conditions that minimize the damage to DNA. Biotinylated DNA is purified with streptavidin beads, in conditions that facilitate association of long DNA filaments. Finally, the DNA is eluted by cleaving the biotin moiety. This approach can enrich over 150-times for replicating DNA and about 50-times in replication fork structures, as verified by EM. This procedure could benefit analysis of replication intermediates in EM as well as other techniques for the study of replicating DNA.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139741403","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Mapping histone variant genomic distribution: Exploiting SNAP-tag labeling to follow the dynamics of incorporation of H3 variants.","authors":"Audrey Forest, Jean-Pierre Quivy, Geneviève Almouzni","doi":"10.1016/bs.mcb.2022.10.007","DOIUrl":"10.1016/bs.mcb.2022.10.007","url":null,"abstract":"<p><p>In the eukaryotic cell nucleus, in addition to the genomic information, chromatin organization provides an additional set of information which is more versatile and associates with distinct cell identities. In particular, the marking of the nucleosomes by a choice of specific histone variants can potentially confer distinct functional properties critical for genome function and stability. To understand how this unique marking operates we need to access to the genomic distribution of each variant. A general approach based on ChIP-Seq, relies on the specific isolation of DNA bound to the variant of interest, usually using cross-linked material and specific antibodies. The availability of reliable specific antibodies recognizing with high affinity crosslinked antigen represents a limitation. Here, we describe an experimental approach exploiting a tag fused to the protein of interest. The chose protein is a histone variant and we use native conditions for the selective capture of the histone variant in a nucleosome. Most importantly, we describe how to use a particular labeling system, with a SNAP tag enabling to specifically capture nucleosomes comprising newly synthesized histones. This method allows to follow the newly deposited histone variant at various times thereby offering a unique opportunity to evaluate the dynamics of histone deposition genome wide. We describe the method here for H3 variant, but it can be adapted to any histone variant with the appropriate fused tag to address genome wide a turn-over associated to the biological context of interest.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139741407","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Methods in cell biologyPub Date : 2024-01-01Epub Date: 2024-04-04DOI: 10.1016/bs.mcb.2024.02.022
J Paul Robinson, Bartek Rajwa
{"title":"Spectral flow cytometry: Fundamentals and future impact.","authors":"J Paul Robinson, Bartek Rajwa","doi":"10.1016/bs.mcb.2024.02.022","DOIUrl":"10.1016/bs.mcb.2024.02.022","url":null,"abstract":"<p><p>Spectral flow cytometry has emerged as a significant player in the cytometry marketplace, with the potential for rapid growth. Despite a slow start, the technology has made significant strides in advancing various areas of single-cell analysis utilized by the scientific community. The integration of spectral cytometry into clinical laboratories and diagnostic processes is currently underway and is expected to garner a significant level of widespread acceptance in the near future. However, incorporating a new methodological approach into existing research programs can lead to misunderstandings or even misuse. This chapter offers an introductory yet comprehensive explanation of the scientific principles that form the foundation of spectral cytometry. Specifically, it delves into the unmixing processes that are utilized for data analysis. This overview is designed for those who are new to the field and seeking an informative guide to this exciting emerging technology.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140863237","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Methods in cell biologyPub Date : 2024-01-01Epub Date: 2024-04-04DOI: 10.1016/bs.mcb.2024.02.012
Kah Teong Soh, Joseph D Tario, Katharine A Muirhead, Paul K Wallace
{"title":"Probing cell proliferation: Considerations for dye selection.","authors":"Kah Teong Soh, Joseph D Tario, Katharine A Muirhead, Paul K Wallace","doi":"10.1016/bs.mcb.2024.02.012","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.02.012","url":null,"abstract":"<p><p>Broadly speaking, cell tracking dyes are fluorescent compounds that bind stably to components on or within the cells so the fate of the labeled cells can be followed. Their staining should be bright and homogeneous without affecting cell function. For purposes of monitoring cell proliferation, each time a cell divides the intensity of cell tracking dye should diminish equally between daughter cells. These dyes can be grouped into two different classes. Protein reactive dyes label cells by reacting covalently but non-selectively with intracellular proteins. Carboxyfluorescein diacetate succinimidyl ester (CFSE) is the prototypic general protein label. Membrane intercalating dyes label cells by partitioning non-selectively and non-covalently within the plasma membrane. The PKH membrane dyes are examples of lipophilic compounds whose chemistry allows for their retention within biological membranes without affecting cellular growth, viability, or proliferation when used properly. Here we provide considerations based for labeling cell lines and peripheral blood mononuclear cells using both classes of dyes. Examples from optimization experiments are presented along with critical aspects of the staining procedures to help mitigate common risks. Of note, we present data where a logarithmically growing cell line is labeled with both a protein dye and a membrane tracking dye to compare dye loss rates over 6days. We found that dual stained cells paralleled dye loss of the corresponding single stained cells. The decrease in fluorescence intensity by protein reactive dyes, however, was more rapid than that with the membrane reactive dyes, indicating the presence of additional division-independent dye loss.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140850284","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Methods in cell biologyPub Date : 2024-01-01Epub Date: 2023-09-11DOI: 10.1016/bs.mcb.2023.05.002
Kiyana Godazandeh, Lies Van Olmen, Lore Van Oudenhove, Steve Lefever, Cedric Bogaert, Bruno Fant
{"title":"Methods behind neoantigen prediction for personalized anticancer vaccines.","authors":"Kiyana Godazandeh, Lies Van Olmen, Lore Van Oudenhove, Steve Lefever, Cedric Bogaert, Bruno Fant","doi":"10.1016/bs.mcb.2023.05.002","DOIUrl":"10.1016/bs.mcb.2023.05.002","url":null,"abstract":"<p><p>Next to conventional cancer therapies, immunotherapies such as immune checkpoint inhibitors have broadened the cancer treatment landscape over the past decades. Recent advances in next generation sequencing and bioinformatics technologies have made it possible to identify a patient's own immunogenic neoantigens. These cancer neoantigens serve as important targets for personalized immunotherapy which has the benefit of being more active and effective in targeting cancer cells. This paper is a step-by-step guide discussing the different analyses and challenges encountered during in-silico neoantigen prediction. The protocol describes all the tools and steps required for the identification of immunogenic neoantigens.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140318635","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Methods in cell biologyPub Date : 2024-01-01Epub Date: 2023-10-11DOI: 10.1016/bs.mcb.2023.05.011
Ting Zhao, Hong Du, Cong Yan
{"title":"Characterization of lysosomal acid lipase in Ly6G<sup>+</sup> and CD11c<sup>+</sup> myeloid-derived suppressor cells.","authors":"Ting Zhao, Hong Du, Cong Yan","doi":"10.1016/bs.mcb.2023.05.011","DOIUrl":"10.1016/bs.mcb.2023.05.011","url":null,"abstract":"<p><p>Lysosomal acid lipase (LAL) is a key enzyme in the metabolic pathway of neutral lipids, whose deficiency (LAL-D) induces the differentiation of myeloid lineage cells into myeloid-derived suppressor cells (MDSCs), which promotes tumor growth and metastasis. This protocol provides detailed procedures for assessment of various LAL biochemical and physiological activities in Ly6G<sup>+</sup> and CD11c<sup>+</sup> MDSCs, including isolation of Ly6G<sup>+</sup> and CD11c<sup>+</sup> cells from the bone marrow and blood of mice, assays of LAL-D-induced cellular metabolic and mitochondrial activities, assessment of LAL-D-induced pathogenic immunosuppressive activity and tumor stimulatory activity. Pharmacological inhibition of the LAL activity was also described in both murine myeloid cells and human white blood cells.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140329971","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}