mammalian Protocols

Category: Microscopy Protocols: Live-Cell Imaging Protocols

This protocol describes a sealed preparation that allows the continuous long-term observation of cultured mammalian cells on upright or inverted microscopes without environmental CO2 control. The preparation allows for optical conditions consistent with high-quality imaging and good cell viability for at least 100 hours. - [Read A Sealed Preparation for Long-Term Observations of Cultured Cells]

Category: PCR Protocols: cDNA Synthesis Protocols

An oligodeoxynucleotide primer hybridized to mRNA is extended by an RNA-dependent DNA polymerase to create a cDNA copy that can be amplified by PCR.  Depending on the purpose of the experiment, the primer for first-strand cDNA synthesis can be specifically designed to hybridize to a particular target gene, or a general primer such as oligo(dT) can be used to prime cDNA synthesis from essentially all mammalian mRNAs - [Read Amplification of cDNA Generated by Reverse Transcription of mRNA Protocol]

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]

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]

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]

Category: Cell Culture Protocols: Sterile Technique Protocols

Cultured mammalian cells are used extensively in cell biology studies; it requires a number of special skills in order to be able to preserve the structure, function, behavior and biology of the cells. This unit describes the basic skills required to maintain and preserve cell cultures: aseptic technique, medium characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. - [Read Basic Techniques for Mammalian Cell Tissue Culture Protocol]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

Cultured mammalian cells are used extensively in cell biology studies; it requires a number of special skills in order to be able to preserve the structure, function, behavior and biology of the cells. This unit describes the basic skills required to maintain and preserve cell cultures: aseptic technique, medium characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. - [Read Basic Techniques for Mammalian Cell Tissue Culture Protocol]

Category: Cell Biology and Cell Culture

Techniques on how to create gradients of iodixanol for the fractionation of mammalian cells. These gradients can be generated as pre-formed discontinuous or continuous gradients. These gradients are invariably run in swinging-bucket rotors in low-speed centrifuges. - [Read C2 Preparation of pre-formed iodixanol gradients for mammalian cells.]

Category: Microinjection Protocols: Microinjection Equipment

This protocol describes an easy method for calibrating micropipette tips that have been pulled in the laboratory. It is essential to estimate the internal diameter of the pulled micropipette tip when adjusting parameters for a new puller or new type of glass tubing. A tip diameter of ~0.3 µm is optimal for the microinjection of mammalian cells in culture (e.g., CHO, PtK1, and COS-7). A 10% increase in diameter increases the delivery rate by more than 30% and can cause cell damage. - [Read Calibration of Micropipette Tips Protocols]

Category: DNA Protocols: Genomic DNA Library Protocols

This stage summarizes how to construct a cDNA library in a mammalian vector, using commercial kits. These kits are available from several manufacturers and generally include all the required reagents. - [Read Construction of cDNA Libraries in Eukaryotic Expression Vectors for Construction and Screening]

Category: Reporter Gene Assays: Luciferase Assay Protocols

It is possible that some cell lines have lost the ability to perform RNAi or that cells derived from certain tissues do not support RNAi. This reporter assay, for RNAi in mammalian cells, can be used to establish whether the cells under study are susceptible to RNAi. - [Read Cotransfection of Luciferase Reporter Plasmids with siRNA Duplexes Protocol]

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

This reporter assay, for RNAi in mammalian cells, can be used to establish whether the cells under study are susceptible to RNAi. - [Read Cotransfection of Luciferase Reporter Plasmids with siRNA Duplexes Protocol]

Category: Cell Culture Protocols: Cell Storing and Cell Freezing Protocols

Protocol provides methods for cryofreezing and subsequent thawing of mammalian cells. Pre-confluent cells are trypsinized, pelleted, resuspended in freezing medium, and gradually frozen. When needed, frozen cells are thawed quickly under running tap water and transferred to growth medium. - [Read Cryopreservation of Mammalian Culture Cells: Preparation and Recovery of Samples Protocol]

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

Protocol describes a denaturing immunoprecipitation (IP) for mammalian cells. Prefer to use denaturing IPs to recover labeled proteins from pulse-chase experiments. The use of denaturing IPs reduces background considerably. - [Read Denaturing Protein Immunoprecipitation from Mammalian Cells Protocol]

Category: Cell Culture Protocols: Cell Culture Contamination Prevention and Removal Protocols

Protocol describes a method for mycoplasma detection using Hoechst 33258, a fluorescent dye that stains DNA. Staining of particulate DNA matter around the cell nucleus (on the cell surface or in the cytoplasm) is indicative of mycoplasma contamination. - [Read Detection of Mycoplasma in Mammalian Cell Cultures Using Fluorescence Microscopy Protocol]

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

Protocol describes the use of western blotting to analyze the RNAi-induced depletion of target protein from mammalian cells.  Immunofluorescence can be used as an alternative. - [Read Detection of RNAi-Induced Protein Knockdown in Mammalian Cells by Western Blotting Protocol]

Category: Western Blot Protocols

Protocol describes the use of western blotting to analyze the RNAi-induced depletion of target protein from mammalian cells.  Immunofluorescence can be used as an alternative. - [Read Detection of RNAi-Induced Protein Knockdown in Mammalian Cells by Western Blotting Protocol]

Category: Gene Delivery Protocols: Biolistics Protocols

In this protocol, gold or tungsten particles are coated with DNA and then shot from a gun into monolayers of mammalian cells. - [Read DNA Transfection by Biolistics Protocol]

Category: Reporter Gene Assays

Protocol describing ecdysone as regulator of inducible gene expression in mammalian cells. - [Read Ecdysone as Regulator of Inducible Gene Expression in Mammalian Cells]

Category: Cell Biology and Cell Culture: Cell Electroporation Protocols

Electroporation of Cell Lines With DNA. Electroporation for the efficient transfection of mammalian cells with DNA. Chu et al. Cellular Immunology Oxford - [Read Electroporation of Cell Lines With DNA]

Category: Cell Culture Protocols

Protocol describes a method for estimation of mammalian cell number in a defined volume of medium using a hemocytometer. Automated methods using cell-counting devices such as those produced by Coulter are desirable when large numbers of individual samples are to be counted. - [Read Estimation of Cell Number by Hemocytometry Counting Protocol]

Category: DNA Protocols: Genomic DNA Library Protocols

Protocol describes how to construct and amplify a genomic DNA library in a plasmid vector (pSPL3) equipped to express cloned exons in transfected mammalian cells. - [Read Exon Trapping and Amplification for Construction of the Library Protocol]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

Acidocalcisomes, the dense acidic calcium-storing organelles, which were originally identified in Trypanosoma cruzi, have no parallels in mammalian cells. They thus represent a unique functional characteristic, not shared by the host and hence offer an important potential target for chemotherapy of Chagas disease. - [Read Fractionation of Acidocalcisomes and Other Organelles from Trypanosoma, Leishmania, Chlamydomonas]

Category: Cell Biology and Cell Culture: Cell Culture Techniques: Cell Line Preservation Freezing

Freezing and Thawing of Mammalian Cell Lines. For long term storage of myeloma cells, hybridoma cells, T cells, and other mammalian cell lines in liquid nitrogen, and restoring them in culture. Kitto Lab Texas. - [Read Freezing and Thawing of Mammalian Cell Lines]

Category: Cell Culture Protocols: Cell Storing and Cell Freezing Protocols

For long term storage of myeloma cells, hybridoma cells, T cells, and other mammalian cell lines in liquid nitrogen, and restoring them in culture. - [Read Freezing and Thawing of Mammalian Cell Lines Protocol]