Current Protocols in Cell Biology最新文献

Induced pluripotent stem cell (iPSC)-based models are powerful tools to study neurodegenerative diseases such as Parkinson's disease. The differentiation of patient-derived neurons and astrocytes allows investigation of the molecular mechanisms responsible for disease onset and development. In particular, these two cell types can be mono- or co-cultured to study the influence of cell-autonomous and non-cell-autonomous contributors to neurodegenerative diseases. We developed a streamlined procedure to produce high-quality/high-purity cultures of dopaminergic neurons and astrocytes that originate from the same population of midbrain floor-plate progenitors. This unit describes differentiation, quality control, culture parameters, and troubleshooting tips to ensure the highest quality and reproducibility of research results. © 2019 The Authors.

Basic Protocol 1: Differentiation of iPSCs into midbrain-patterned neural progenitor cells

Support Protocol: Quality control of neural progenitor cells

Basic Protocol 2: Differentiation of neural progenitor cells into astrocytes

Basic Protocol 3: Differentiation of neural progenitor cells into dopaminergic neurons

Basic Protocol 4: Co-culture of iPSC-derived neurons and astrocytes

Telomerase plays a critical role in cancer and aging by adding hexa-nucleotide repeats to the ends of telomeres and extending the cellular proliferative lifespan. The very low level of telomerase expression in most cell populations and the difficulty of detecting telomere elongation in single cells have limited the study of telomerase expression and function in individual cells of a heterogeneous population. The method described in this article combines single-molecule detection (RNAscope) of telomerase reverse transcriptase (TERT) with our previously described TSQ1 assay for in situ monitoring of telomere extension, thereby enabling detection of TERT expression, telomere length, and telomere elongation in single cells and providing a unique approach for studying the factors that regulate telomere elongation by telomerase. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: TSQ1 lentivirus production

Basic Protocol 2: TSQ1 lentiviral infection and plating

Basic Protocol 3: RNAscope analysis

Basic Protocol 4: TSQ1 PNA-FISH detection

Cover: In Ioannou et al. (https://doi.org/10.1002/cpcb.95). Immunostaining of astrocytes and microglia after transfer assay. After the transfer assay, glia were fixed and immunostained with anti-GFAP and anti-Iba1 to label astrocytes and microglia, respectively. Cells were imaged using a Zeiss 880 confocal microscope with a 63× objective. Scale bars are 10 εm.

Protein-protein interactions (PPIs) add an essential layer of complexity to the information encoded by the genome. Modulation of such interactions is a key feature of most, if not all, cellular activities and allows cells to respond rapidly to both internal and external signals and stimuli. In this respect, the development of the BioID assay to interrogate PPIs within a cellular context represents an important adjunct to the range of tools currently at researchers' disposal. To address some of its current limitations, we devised 2C-BioID, in which biotin ligase and the protein of interest remain as separate entities until induced to associate. This is accomplished using the well-established FKBP-FRB dimerization system (based on the rapamycin-induced binding of FK506 binding protein and FKBP12-rapamycin binding domain.). The design of 2C-BioID ensures that biotin ligase association with the protein of interest occurs only after addition of the rapamycin analogue AP21967. As such, 2C-BioID alleviates potential targeting issues and improves the ability to exclude false positives, thereby refining the specificity of BioID-generated interactomes. © 2019 by John Wiley & Sons, Inc.

Spatial distribution of chromatin-associated proteins provides invaluable information for understanding gene regulation. Conventional immunostaining is widely used for labeling chromatin-associated proteins in many cell types. However, for a subset of difficult cell types, such as differentiated human keratinocytes, achieving high-quality immunostaining for nuclear proteins remains challenging. To overcome this technical barrier, we developed the nuclei isolation staining (NIS) method. In brief, NIS involves rapid isolation of nuclei from live cells, followed by fixation and staining of the nuclei directly on coverslips for subsequent high-magnification imaging. By removing the cytoplasmic contents and staining just the nuclei, this NIS method drastically improves antibody labeling efficiency for chromatin-associated proteins. In this article, we describe the development and a step-by-step protocol of NIS, using differentiated human keratinocytes as an example. We also discuss other applications, based on the principle of this NIS method, for understanding cell-type and cell-state specific gene regulation. © 2019 by John Wiley & Sons, Inc.

Neurons and glia operate in a highly coordinated fashion in the brain. Although glial cells have long been known to supply lipids to neurons via lipoprotein particles, new evidence reveals that lipid transport between neurons and glia is bidirectional. Here, we describe a co-culture system to study transfer of lipids and lipid-associated proteins from neurons to glia. The assay entails culturing neurons and glia on separate coverslips, pulsing the neurons with fluorescently labeled fatty acids, and then incubating the coverslips together. As astrocytes internalize and store neuron-derived fatty acids in lipid droplets, analyzing the number, size, and fluorescence intensity of lipid droplets containing the fluorescent fatty acids provides an easy and quantifiable measure of fatty acid transport. © 2019 The Authors.

Chromatin-associated proteins are instrumental for controlling spatiotemporal gene expression. Determining where these proteins bind across the genome is critical for understanding gene regulation. A widely used technique at present is ChIP-seq, which leverages chromatin fragmentation, antibody-mediated enrichment, next-generation sequencing, and data analysis to uncover the genomic sequences and patterns of protein-DNA interactions. In this article, we will provide an overview of how ChIP-seq was developed, the key elements of the experimentation and data analysis pipeline, and the recent variations that push the boundaries of precision and cell number requirements. We will also briefly discuss how future development of ChIP-seq may further advance our understanding of chromatin biology. © 2019 by John Wiley & Sons, Inc.

Whether screening small mammal serum during antibody production or attempting to preserve a stock of precious antibody, this protocol's western blotting method using aliquots containing nanoliter volumes of antibody will benefit researchers. Time-tested western blotting workflows allowing separation and analysis of proteins are routinely utilized in clinical and laboratory settings. The necessity for relatively large quantities of antibody is a major limitation to this universal tool. This article provides a step-by-step protocol for detecting proteins of interest with solutions containing nanoliter volumes of antibody without altering the preceding gel electrophoresis and transfer methods. Important considerations, frequently encountered problems, and means of optimizing reproducibility are discussed. Complementary diagrams, images, and videos are provided. The protocol is demonstrated using 0.3 nanoliters of anti-serum to detect fibronectin in a human foreskin fibroblast cell line. Finally, two support protocols detailing methods of extracting proteins from cultured cells are reported. Published 2019. This article is a US Government work and is in the public domain in the USA.

Cover: In Rezanejad et al. (https://doi.org/10.1002/cpcb.82). Intact whole-mount immunostaining of organoids after growth for 14 days. See e82.