nuclei Protocols

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

Simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and proteolytic treatment to permeabilize cells. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells for subsequent culturing based on DNA-content differences, is also described. Also presented are methods for staining of cell nuclei isolated from paraffin-embedded tissues, and deconvolution of DNA-content-frequency... - [Read Analysis of Cellular DNA Content by Flow Cytometry Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

This protocol provides a method for analysis of DNA fragmentation using flow cytometry after propidium iodide (PI) labeling of apoptotic nuclei. Apoptotic nuclei stain less than their normal counterparts because of diffusion of low-molecular-weight fragments out of the cells and/or chromatin condensation. - [Read Analysis of DNA Fragmentation Using Propidium Iodide (PI) Fluorescence of Individual Nuclei Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

This protocol provides a method for analysis of DNA fragmentation using flow cytometry after propidium iodide (PI) labeling of fixed apoptotic cells. Flow cytometry reveals apoptotic nuclei in the subdiploid region of the cell cycle histogram... - [Read Analysis of DNA Fragmentation Using Propidium Iodide (PI) Staining After Ethanol Fixation Protocol]

Category: Cell Organelle Protocols: Nucleus Protocols

The key step is the lysis which solubilizes centrosomes away from nuclei by very low ionic strength lysis after treatment of cells with nocodazole and cytochalasin B. The released centrosomes are then centrifuged onto a Ficoll cushion (to avoid pelleting) and the interface between the lysate and the Ficoll is collected and the centrosomes are concentrated on a sucrose gradient. Fractions are assayed by spindown and double IF with 5051 serum and anti-tubulin and the pooled fractions are frozen... - [Read CHO Centrosome Prep Protocol]

Category: Fungi Protocols: Fungi Genetics Protocols

Standard operating procedure for the determination of tissue fungal burden utilizing quantitative real time polymerase chain reaction (QPCR). This standard operating procedure will provide information on how to assess fungal tissue burden of infected animals by use of a single copy (FKS) or multicopy gene (18s RNA) to assess the number of fungal cell nuclei present. - [Read Determination of Tissue Fungal Burden Utilizing Quantitative Real Time Polymerase Chain Reaction]

Category: DNA Protocols: DNA Extraction Blood

Purifying nuclear pellets, Extraction of DNA from nuclei. Method for DNA isolation from Blood. Genomic Variation Laboratory, UC Davis. - [Read DNA extraction from blood]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Protocol for fluorescence in situ hybridization (FISH) on human chromosomes and interphase nuclei. Includes: Plasmid probes; COSMID-, YAC- and LIBRARY PROBES; Probe detection. - [Read Fluorescence In Situ Hybridization (FISH) on Human Chromosomes and Interphase Nuclei Protocol]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to obtain naked nuclei extruded from the cell. - [Read How to Obtain Naked Nuclei Extruded From the Cell]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to stain and count nuclei. - [Read How to Stain and Count Nuclei]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Hybridisation of Interphase Nuclei Method. Sanger Institute. - [Read Hybridisation of Interphase Nuclei Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

To accurately predict the activity of a transgene it is critical to understand its location and dynamics in the 3-D interphase nucleus. Developed in situ methods to visualize transgenes (including single copy genes) & their transcripts during interphase from different tissues & plant species. These techniques reduce the time necessary for characterization of transgene integration by eliminating the need for time-consuming segregation analysis and extend characterization to the interphase nucleus - [Read In Situ Methods to Localize Transgenes and Transcripts in Interphase Nuclei]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

In situ methods to visualize transgenes (including single copy genes) and their transcripts during interphase from different tissues and plant species. These techniques reduce the time necessary for characterization of transgene integration by eliminating the need for time-consuming segregation analysis, and extend characterization to the interphase nucleus, thus increasing the likelihood of accurate prediction of transgene activity. - [Read In Situ Methods to Localize Transgenes and Transcripts in Interphase Nuclei]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Interphase nuclei preparation for FISH. Sanger Institute. - [Read Interphase nuclei]

Category: Tetrahymena Protozoa Protocols

Protocol describes how to allow the isolation of nuclei from all stages of the Tetrahymena life cycle in high yield with a high degree of purity. This method gives highly purified populations of both micronuclei and macronuclei. - [Read Isolation and Purification of Tetrahymena Nuclei Protocol]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Protocol for the isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays. Plant materials are ground in hexylene glycol-based buffers and highly enriched nuclear fractions are obtained using Percoll density gradients. Standard and small-scale protocols are presented, along with a tested method for nuclear run-on assays. The entire process may be completed within 3 days. - [Read Isolation of Arabidopsis Nuclei and Measurement of Gene Transcription Rates Protocol]

Category: Cell Organelle Protocols: Nucleus Protocols

Protocol for the isolation of interphase nuclei from Paraffin Section for FISH using the Hedley Technique. - [Read Isolation of Interphase Nuclei from Paraffin Section for FISH, Hedley Technique]

Category: Protein Protocols: Nuclear Extraction Protocols

Homogenize cells or tissue, Pellet nuclei, Lyse nuclei, Reprecipitate nuclear proteins, dialyze nuclear extract. Hattori M et al., DNA Cell Biol. 1990. Homogenization buffer and dialysis buffer recipes. - [Read Large scale nuclear extract preparation]

Category: DNA Protein Interactions Protocols: Dnase Footprinting Assay Protocols

Protocol describes how isolated nuclei are incubated with varying amounts of Dnase I. Genomic DNA is then isolated from the nuclei and digested with a restriction enzyme, analyzed by gel electrophoresis, and probed by Southern hybridization. - [Read Mapping Dnase-I-hypersensitive Sites Protocol]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

Protocol for nuclei isolation. - [Read Nuclei Isolation Protocol]

Category: Cytogenetics Protocols

An ideal method of tissue preparation ensures both good specimen morphology and that the target molecules are in the optimum state for probe access and hybridization. DNA:DNA in situ hybridization is usually carried out on chromosome spread preparations where chromosome and nuclei are released from cells and spread on a glass microscope slide. This method yields well separated and enlarged chromosomes with good morphology which can be analyzed in transmitted light or fluorescence microscopes. - [Read Preparation of Chromosome Spreads]

Category: Xenopus Protocols: Xenopus Egg Extracts Protocols

Protocol describes the preparation of nuclei from Xenopus sperm. These nuclei can be used for spindle assembly assays, microscopy, or DNA synthesis. - [Read Preparation of Xenopus Sperm Nuclei Protocol]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes a useful way to observe the development of embryos, as well as meristems & young primordia developing at the shoot apex by confocal microscopy after staining the nuclei with propidium iodide. The number of cells can be exactly quantified in a meristem or in young primordia. Because embryonic & meristematic cells are largely filled out by their nuclei, it is easier to image only the nuclei. This method allows analysis of whole-mount material, which is more easily reconstructed. - [Read Protocol for Nuclear Staining of Plants for Confocal Microscopy]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

In this protocol nuclei isolated from cells expressing the gene of interest are incubated with radiolabeled UTP which is incorporated into nascent RNA transcripts by RNA polymerase molecules that were actively transcribing at the time the cells were harvested. Because very little denovo initiation of RNA synthesis occurs in isolated nuclei, transcription of the target gene can be measured by hybridizing the radiolabeled RNA to an excess of the target gene immobilized on a nitrocellulose or nylon - [Read Protocol for Transcriptional Run- On Assays]

Category: Cell Biology and Cell Culture: Endothelial Cell Protocols

Primary mammalian endothelial cells protocol. This protocol is designed for primary endothelial cells isolated from various organs of mammals. Large and flat cells, often with large nuclei. Includes: Required reagents; DNA preparation and quality; Preparation of cells and cell culture; Important controls; Nucleofection protocol. - [Read Protocol Primary Mammalian Endothelial Cells]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

This protocol describes an in vitro reaction to assay mitotic spindle assembly. The assay uses Cytostatic Factor extract made from Xenopus eggs, fluorescently-labelled tubulin, and prepared sperm nuclei. Spindle assembly is monitored by immunofluorescence microscopy. - [Read Protocol Spindle Assembly In Vitro]