Springer protocols feed by Cell Biology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Global Run-On Sequencing (GRO-seq) Library Preparation from Drosophila Ovaries
In the past decade, deep-sequencing approaches have greatly improved our knowledge of the genome’s potential and have become a crucial milestone for new discoveries in genomics. Transcription is the first step of gene expression; therefore, the detection and measurement of transcription rates is of great interest. Here, a detailed protocol for global run-on sequencing (GRO-seq) library preparation from Drosophila ovaries is described. The method relies on rapid isolation of nuclei with halted transcription, then restarting transcription in physiological conditions in the presence of a labeled nucleotide. The newly tr...
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
Analysis of Cell Cycle Switches in Drosophila Oogenesis
The study of Drosophila oogenesis provides invaluable information about signaling pathway regulation and cell cycle programming. During Drosophila oogenesis, a string of egg chambers in each ovariole progressively develops toward maturity. Egg chamber development consists of 14 stages. From stage 1 to stage 6 (mitotic cycle), main-body follicle cells undergo mitotic divisions. From stage 7 to stage 10a (endocycle), follicle cells cease mitosis but continue three rounds of endoreduplication. From stage 10b to stage 13 (gene amplification), instead of whole genome duplication, follicle cells selectively amplify specific geno...
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
Detection of Cell Death and Phagocytosis in the Drosophila Ovary
Billions of cells die and are cleared throughout the development and homeostasis of an organism. Either improper death or clearance can lead to serious illnesses. In the adult Drosophila ovary, germline cells can die by programmed cell death (PCD) at three distinct stages; here we focus on cell death that occurs in mid- and late oogenesis. In mid-oogenesis, the germline of egg chambers can undergo apoptosis in response to nutrient deprivation. In late oogenesis, the nurse cells are eliminated through a developmentally regulated, non-apoptotic cell death. In this chapter, we describe several methods to detect cell death and...
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
Visualizing Cytoophidia Expression in Drosophila Follicle Cells via Immunohistochemistry
We describe a user-friendly immunohistochemical approach for the detection of protein localization in Drosophila ovaries, here focusing on CTP synthase. This approach mainly uses fluorescently labeled antibodies to detect single, double, or multiple antigens. We provide a step-by-step protocol with detailed notes and tips, a simplified method that can also be adapted to detect protein localization beyond Drosophila ovaries. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
Immuno-Electron Microscopy and Electron Microscopic In Situ Hybridization for Visualizing piRNA Biogenesis Bodies in Drosophila Ovaries
Immuno-electron microscopy and electron microscopic in situ hybridization are powerful tools to identify the precise subcellular localization of specific proteins and RNAs at the ultramicroscopic level. Here we describe detailed procedures for how to detect the precise location of a specific target labeled with both fluorescence and gold particles. Although they have been developed for the analysis of Drosophila ovarian somatic cells, these techniques are suitable for a wide range of biological applications including human, primate, and rodent analysis. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
Ultrastructural Analysis of Drosophila Ovaries by Electron Microscopy
The Drosophila melanogaster ovary is a powerful, genetically tractable system through which one can elucidate the principles underlying cellular function and organogenesis in vivo. In order to understand the intricate process of oogenesis at the subcellular level, microscopic analysis with the highest possible resolution is required. In this chapter, we describe the preparation of ovaries for ultrastructural analysis using transmission electron microscopy and focused ion beam scanning electron microscopy. We discuss and provide protocols for chemical fixation of Drosophila ovaries that facilitate optimal imaging with parti...
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
Fluorescent In Situ Hybridization of Nuclear Bodies in Drosophila melanogaster Ovaries
Fluorescent in situ hybridization (FISH) is a technique for determining the cytological localization of RNA or DNA molecules. There are many approaches available for generating in situ hybridization probes and conducting the subsequent hybridization steps. Here, we describe a simple and reliable FISH method to label small RNAs (200–500 nucleotides in length) that are enriched in nuclear bodies in Drosophila melanogaster ovaries, such as Cajal bodies (CBs) and histone locus bodies (HLBs). This technique can also be applied to other Drosophila tissues, and to abundant mRNAs such as histone transcripts. (Source: Springe...
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
Cell Cycle Dynamics of Proteins and Post-translational Modifications Using Quantitative Immunofluorescence
Immunofluorescence can be a powerful tool to detect protein levels, intracellular localization, and post-translational modifications. However, standard immunofluorescence provides only a still picture and thus lacks temporal information. Here, we describe a method to extract temporal information from immunofluorescence images of fixed cells. In addition, we provide an optional protocol that uses micropatterns, which increases the accuracy of the method. These methods allow assessing how protein levels, intracellular localization, and post-translational modifications change through the cell cycle. (Source: Springer protocol...
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
Spatiotemporal Investigation of Phosphorylation Events During Cell Cycle Progression
Polo-like kinase 1 (Plk1) is an essential kinase for mitotic commitment and progression through mitosis. In contrast to its well characterized roles during mitosis, the precise molecular events controlled by Plk1 during G2/M progression and their spatiotemporal regulation are still poorly elucidated. We recently investigated Plk1-dependent regulation of Cdc25C phosphatase, an activator of the master mitotic driver Cyclin B1-Cdk1. To this end, we generated a genetically encoded FRET (Förster Resonance Energy Transfer)-based Cdc25C phosphorylation biosensor to observe Cdc25 spatiotemporal phosphorylation during cell cyc...
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
Elutriation for Cell Cycle Synchronization in Fission Yeast
Cell synchronization is a powerful technique for studying the eukaryotic cell cycle events precisely. The fission yeast is a rod-shaped cell whose growth is coordinated with the cell cycle. Monitoring the cellular growth of fission yeast is a relatively simple way to measure the cell cycle stage of a cell. Here, we describe a detailed method of unperturbed cell synchronization, named centrifugal elutriation, for fission yeast. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
Cell Cycle Synchronization in Xenopus Egg Extracts
Many important discoveries in cell cycle research have been made using cell-free extracts prepared from the eggs of the South African clawed frog Xenopus laevis. These extracts efficiently support the key nuclear functions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. Here, we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei. We d...
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
Cell Synchronization of Mouse Embryonic Fibroblasts
A fundamental need in the analysis of the cell cycle is the ability to isolate relatively homogeneous populations of cells in different phases. This is complicated by the variable proliferative properties and responses to synchronizing methods of different cancer-derived cell lines. Paradoxically, cell lines with genetic defects in cell cycle control are sometimes chosen because they are amenable to chemical synchronization. Embryonic fibroblasts from mice present the opportunity to study the effects of defined genetic modifications on a normal cell cycle. However, synchronization of these cells has often been challenging....
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
Measurement of Cdk1/Cyclin B Kinase Activity by Specific Antibodies and Western Blotting
We present a method to measure the activity of the enzyme Cdk1/cyclin B. This enzyme is required by all eukaryotic cells to enter mitosis. Therefore, a biochemical assay to measure Cdk1/cyclin B activity can be used to identify cell populations that are in mitosis or to detect inhibitors of Cdk1/Cyclin B in vitro. A key distinction of the method presented here, compared to others, is that it uses a recombinant protein, a specific antibody, and a western blot apparatus, which makes the technique available to cell and molecular biology laboratories who do not wish to use radioisotopes, which are commonly required for other p...
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
Imaging Cell Cycle Phases and Transitions of Living Cells from Yeast to Woman
We present the different approaches, review different types of reporters, and discuss in depth all the aspects to be considered to obtain optimal results. We also describe our latest cell cycle markers, which afford unprecedented “sub”-phase temporal resolution. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
Using the Fly-FUCCI System for the Live Analysis of Cell Cycle Dynamics in Cultured Drosophila Cells
Cultured Drosophila cells are an attractive system for live imaging experiments, as this cell type is not very demanding in terms of temperature and media composition. Moreover, cultured Drosophila cell lines are very responsive to RNAi without being prone to off-target effects, and thus have become important for use in high-content screening. We have developed a fly-specific fluorescent, ubiquitination-based cell cycle indicator (FUCCI) system that enables faithful detection of G1, S, and G2 phases, and is thus a powerful tool for the analysis of cell cycle dynamics in living or fixed cells. Here, we describe a protocol f...
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
