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Analysis of Cellular DNA Content by Flow Cytometry Protocol - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E661C8A9C23374B5FAB0610666CCDFB&objectid=6674F58BDEF6DCA5276258FEA4CD534D

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]

Analysis of Clones for Exon Trapping and Amplification - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3651

Category: DNA Protocols: Genomic DNA Library Protocols

Amplification and sequencing of the exon-trapped products. - [Read Analysis of Clones for Exon Trapping and Amplification]

Analysis of DNA Content and Surface Immunophenotype Protocol Using Ethanol Fixation - http://www.cyto.purdue.edu/flowcyt/research/cytotech/telford/ethanol.htm

Category: Immunohistochemistry Protocols: Fixation Protocols

Protocol for analysis of DNA content and surface immunophenotype using gentle ethanol fixation techniques. - [Read Analysis of DNA Content and Surface Immunophenotype Protocol Using Ethanol Fixation]

Analysis of DNA Fragmentation Using Agarose Gel Electrophoresis (Subscription Required) - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4429

Category: Cell Biology and Cell Culture: Apoptosis Protocols: Apoptosis Analysis

Analysis of DNA Fragmentation Using Agarose Gel Electrophoresis Shailaja Kasibhatla et al. This protocol provides a qualitative method for assessing cell death by detecting DNA fragments using agarose gel electrophoresis. One of the classic features of apoptosis is the cleavage of the genomic DNA into oligonucleosomal fragments represented by multiples of 180-200 bp. Visualizing these fragments can aid in characterizing an apoptotic event. May be combined with more quantitative methods. - [Read Analysis of DNA Fragmentation Using Agarose Gel Electrophoresis (Subscription Required)]

Analysis of DNA Fragmentation Using Propidium Iodide (PI) Fluorescence of Individual Nuclei Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4430

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]

Analysis of DNA Fragmentation Using Propidium Iodide (PI) Staining After Ethanol Fixation Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4431

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]

Analysis Of DNA Fragmentation Using The JAM Assay (Subscription Required) - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4432

Category: Cell Biology and Cell Culture: Apoptosis Protocols: Apoptosis Analysis

Analysis of DNA Fragmentation Using the JAM Assay. By Shailaja Kasibhatla et al., The JAM assay is based on labeling nuclear DNA of cycling cells with [3H]thymidine and harvesting samples on glass fiber filters. Apoptosis will generate DNA fragments small enough to pass through the glass fiber filter, resulting in decreased radioactivity of the particular sample. Cell-mediated cytotoxicity or cell killing mediated by cytotoxic T lymphocytes (CTL) can also be measured by this technique. - [Read Analysis Of DNA Fragmentation Using The JAM Assay (Subscription Required)]

Analysis of Recombinant Bacteriophage M13 Clones Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3996

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

A rapid method to analyze the size of the single-stranded DNA of M13 recombinants. - [Read Analysis of Recombinant Bacteriophage M13 Clones Protocol]

Analyzing Yeast Colonies by PCR Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4015

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast PCR Protocols

Yeast colonies are suspended in complete PCR buffer and transferred to a thermal cycler for 35 cycles of PCR. The products of the amplification reaction are analyzed by gel electrophoresis. - [Read Analyzing Yeast Colonies by PCR Protocol]

Annealing siRNAs to Produce siRNA Duplexes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4340

Category: RNAi Technology Protocols

Protocol describes a procedure to anneal sense and antisense siRNA strands to form a duplex. The duplexes can then be transfected into cultured cells. - [Read Annealing siRNAs to Produce siRNA Duplexes Protocol]

Annexin V Protocol for Flow Cytometry - http://www.niehs.nih.gov/research/atniehs/core/fc/protocols/annexin.cfm

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Different cell types vary in their phosphatidylserine (PS) content, along with the amount of PS exposure on the cell surface after cell death. This protocol is a guideline for getting started, however it may be necessary to adjust the concentration of the Annexin V-FITC. - [Read Annexin V Protocol for Flow Cytometry]

Antibody Addition and Detection for Staining Caenorhabditis elegans Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4523

Category: C. elegans Protocols: C. elegans Staining Protocols

Extreme care should be used to identify and verify positive reactions, however, because cross-reactions are common. Counterstaining is essential for examining worms by immunofluorescence and is used to identify the exact cell in which an antigen appears. Methods for counterstaining include labeling all cells with a fluorescent dye that is specific for nucleic acids (e.g., DAPI or propidium iodide) and using GFP driven by tissue-specific promoters. - [Read Antibody Addition and Detection for Staining Caenorhabditis elegans Protocol]

Antibody Addition to Drosophila Specimens and Detection Using Enzyme-Linked Reagents Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4526

Category: Drosophila Protocols

Enzyme-linked reagents give excellent sensitivity and use a simple light microscope for detection. A range of enzymes is available, but for staining in situ, horseradish peroxidase will suit most needs. Diaminobenzidine (DAB) is one of the most sensitive substrates for horseradish peroxidase. It yields an intense brown product that is insoluble in both water and alcohol. It can be made more sensitive by adding metal salts such as cobalt or nickel to the substrate solution. - [Read Antibody Addition to Drosophila Specimens and Detection Using Enzyme-Linked Reagents Protocol]

Antibody Addition to Drosophila Specimens and Detection Using Fluorochrome-Linked Reagents Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4527

Category: Drosophila Protocols

Protocol for antibody addition to Drosophila specimens and detection using fluorochrome-linked reagents. Fluorochrome-linked reagents should be used when high resolution is needed or if two antigens need to be localized simultaneously. Because of the thickness of fly specimens, detection requires access to a confocal microscope. - [Read Antibody Addition to Drosophila Specimens and Detection Using Fluorochrome-Linked Reagents Protocol]

Assay for ß-galactosidase in Extracts of Mammalian Cells - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3952

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

The assay for ß-galactosidase relies on the ability of the enzyme to catalyze the hydrolysis of ONPG (o-nitrophenyl-ß-D- galactopyranoside) to free o-nitrophenol, which absorbs light at 420 nm. In this protocol, extracts of cells transfected with a ß-galactosidase reporter plasmid are incubated with ONPG. - [Read Assay for ß-galactosidase in Extracts of Mammalian Cells]

Assay for ß-galactosidase in Extracts of Mammalian Cells Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3952

Category: Yeast Protocols: Yeast Genetics Protocols: B-Galactosidase Assay Protocols

In this protocol, extracts of cells transfected with a ß-galactosidase reporter plasmid are incubated with ONPG. - [Read Assay for ß-galactosidase in Extracts of Mammalian Cells Protocol]

Assay for Luciferase in Extracts of Mammalian Cells Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3642

Category: Reporter Gene Assays: Luciferase Assay Protocols

In this protocol, cells transfected with a luciferase reporter plasmid are lysed in a detergent-containing buffer. Luciferase in the extract catalyzes an oxidation reaction in which D-luciferin is converted to oxyluciferin, with production of light at 556 nm that can be quantified in a luminometer. - [Read Assay for Luciferase in Extracts of Mammalian Cells Protocol]

Assay of ß-Galactosidase in Yeast: Assay of Crude Extracts - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4157

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

Method describes how a crude extract is prepared, and the activity is normalized to the amount of protein assayed. This method is particularly suitable for comparing cells that are grown under very different conditions or that have different genetic backgrounds. - [Read Assay of ß-Galactosidase in Yeast: Assay of Crude Extracts]

Assay of ß-Galactosidase in Yeast: Assay of Crude Extracts Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4157

Category: Yeast Protocols: Yeast Genetics Protocols: B-Galactosidase Assay Protocols

Protocol describes how a crude extract is prepared, and the activity is normalized to the amount of protein assayed. This method is particularly suitable for comparing cells that are grown under very different conditions or that have different genetic backgrounds. - [Read Assay of ß-Galactosidase in Yeast: Assay of Crude Extracts Protocol]

Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in 96-Well Plates - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4160

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

Method described indicates how cells are lysed by a simple freeze/thaw protocol and treated with a chemiluminescent substrate. - [Read Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in 96-Well Plates]

Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in 96-Well Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4160

Category: Yeast Protocols: Yeast Genetics Protocols: B-Galactosidase Assay Protocols

In this protocol the cells are lysed by a simple freeze/thaw protocol and treated with a chemiluminescent substrate. - [Read Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in 96-Well Protocol]

Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in Microcentrifuge Tubes - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4159

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

In the method described here, the cells are lysed by a simple freeze/thaw protocol and . . . - [Read Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in Microcentrifuge Tubes]

Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in Microcentrifuge Tubes - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4159

Category: Yeast Protocols: Yeast Genetics Protocols: B-Galactosidase Assay Protocols

In this protocol the cells are lysed by a simple freeze/thaw protocol. - [Read Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in Microcentrifuge Tubes]

Assay of ß-Galactosidase in Yeast: Permeabilized Cell Assay - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4158

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

In the method described here, the cells are permeabilized to allow the substrate to enter the cells and the activity is normalized to the number of cells assayed. - [Read Assay of ß-Galactosidase in Yeast: Permeabilized Cell Assay]

Assay of ß-Galactosidase in Yeast: Permeabilized Cell Assay Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4158

Category: Yeast Protocols: Yeast Genetics Protocols: B-Galactosidase Assay Protocols

In this protocol, the cells are permeabilized to allow the substrate to enter the cells and the activity is normalized to the number of cells assayed. - [Read Assay of ß-Galactosidase in Yeast: Permeabilized Cell Assay Protocol]

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In Vitro Translated Xenopus Mos Kinase Assay Protocol

In Vitro Translated Xenopus Mos Kinase Assay Protocol. In response to progesterone, immature Xenopus oocytes mature to eggs that can be fertilized. The Mos protein kinase is essential for oocyte maturation, most likely due to its ability to activate the MAP kinase cascade. This MAP kinase cascade eventually leads to the activation of Cdc2/cyclin B and entry into M phase. In this protocol, tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay.

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