derived Protocols

Category: PCR Protocols: cDNA Synthesis Protocols

To generate "3'-end" partial cDNA clones, mRNA is reverse-transcribed using a "hybrid" primer (Qtotal, QT) that consists of two mixed bases (GATC/GAC followed by [T]17) and a unique 35-base oligonucleotide sequence (QI-QO).  Amplification is then performed using a primer containing part of this sequence (Qouter, Qo) (which now binds to each cDNA at its 3'-end) and a primer derived from the gene of interest, GSP1 (gene-specific primer 1). - [Read 3'-End cDNA Amplification Using Classic RACE Protocol]

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants. - [Read A Novel Fluorescent pH Probe for Expression in Plants]

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

Protocol derived from a paper by Miriam Braunstein and is based on work from the Allis lab. - [Read CHROMATIN IMMUNOPRECIPITATION (CHIP) PROTOCOL FOR YEAST PDF]

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: Stem Cell Protocols: Stem Cell Culture Protocols

Culture conditions have been established for a second blastocyst-derived cell line, trophoblast stem (TS) cells, in addition to embryonic stem (ES) cells. This protocol describes a method for culturing TS cell lines. These cells can then be used to study trophoblast differentiation and placental function. - [Read Culturing Trophoblast Stem (TS) Cell Lines Protocol]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

Protocol for isolation of dendritic cell derived exosomes. - [Read Dendritic Cell Derived-Exosomes]

Category: PCR Protocols

Method uses PCR to amplify and display many cDNAs derived from the mRNAs of a given cell or tissue type.  The method relies on two different types of synthetic oligonucleotides: anchored antisense primers and arbitrary sense primers.  A typical anchored primer is complementary to approx. 13 nucleotides of the poly(A) tail of mRNA and the adjacent two nucleotides of the transcribed sequence. - [Read Differential Display-PCR Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Liver Toxicity Assays Protocols

This test is designed to detect irreversible toxic effects on both cell growth and survival, by the evaluation of colony-forming (CF) efficiency, in hepatoma cell lines derived from man, rat and mouse. - [Read Hepatoma Cell Cultures as In Vitro Models for Hepatotoxicity]

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

This protocol describes the use of SEMV cells (a cell line derived from ram seminal vesicles) in studies into prostaglandin H synthase-mediated metabolism of xenobiotics in intact cells. - [Read In Vitro Model For Studies Of Prostagland H Synthase (PHS)- Mediated Genotoxicity Of Xenobiotics]

Category: Immunology: Mononuclear Cell Isolation Protocols

Mononuclear phagocyte progenitor cells derived from femoral and tibial bone marrow are propagated in the presence of M-CSF. This macrophage growth factor is secreted by L929 cells and is used in the form of L929 cell conditioned medium. The progenitor cells proliferate and differentiate through monoblast, promonocyte and monocyte stages before maturing to macrophages. At this time the cells become firmly adherent to the culture vessel. - [Read Isolation and Culture of Mouse Bone Marrow-Derived Macrophages Protocol]

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

This procedure describes the isolation and culture of adult mouse cardiac myocytes from two or more hearts. Includes modifications for the digestion of two or more hearts in the same procedure and subsequent pooling of myocytes derived from the multiple hearts. The isolation procedure is performed by one or more technicians and routinely yields approximately 1 million rod-shaped myocytes per heart. - [Read Isolation of Adult Mouse Cardiac Myocytes from Two or More Hearts Protocol]

Category: Mouse Protocols

Protocol that investigates macrophage activation using a more homogeneous population of cells, macrophages derived from immature progenitor cells in bone marrow can be studied. A second protocol describes the isolation of bone marrow-derived progenitor cells and propagation of these immature macrophages by CSFs or IL-3. - [Read Isolation of Murine Macrophages Protocol]

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, etc. Protocol provides a thorough description of LCM techniques, with an emphasis on tips and troubleshooting advice derived from LCM users. The total time required to carry out this protocol is typically 1–1.5 h. - [Read Laser-capture Microdissection Protocol]

Category: DNA Protein Interactions Protocols: Dnase Footprinting Assay Protocols

Dnase I is used to fragment a radiolabeled target DNA in the presence and absence of a nuclear extract.  A "footprint" is generated when a protein binds to the target and protects a specific segment of DNA from the nucleolytic activity of Dnase I. By comparing the electrophoretic mobility of the Dnase I cleavage products to those of a sequence ladder derived from the same DNA fragment, the position(s) of the DNA sequences recognized by DNA-binding proteins can be determined. - [Read Mapping Protein-binding Sites on DNA by Dnase I Footprinting Protocol]

Category: Cell Culture Protocols: Cell Lines Protocols

RAW 264.7 cells are a macrophage-like, Abelson leukemia virus transformed cell line derived from BALB/c mice. For routine maintenance in culture (passage), cells are seeded at a confluence of approximately 10% (1 x 106 and 3 x 106 cells in 100-mm and 150-mm plates, respectively) and grown to a confluence of approximately 80%. This procedure requires the cells to be split every two days. - [Read Passage Procedure for RAW 264.7 Cells]

Category: Viral Manipulation Protocols: Phage Protocols

Protocol describes a method for generating isolated plaques from a stock of bacteriophage lambda. Each plaque derives from infection of a single bacterium by a single bacteriophage particle. Because each plaque contains the progeny of a single virus particle, the bacteriophages derived from a single plaque are essentially genetically identical to one another. - [Read Plating Bacteriophage Lambda Protocol]

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

Protocol for the production of completely ES cell-derived fetuses by aggregation with tetraploid embryos. Includes: Recovery of 2-cell stage embryos; Production of tetraploid embryos; Preparation of aggregation plate; Removal of Zona Pellucida; ES cells/ tetraploid embryo "SANDWICH" aggregation; Transfer of embryos. - [Read Production of Completely ES Cell-Derived Fetuses by Aggregation with Tetraploid Embryos]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

This protocol is employed for the purification of a population of muscle-derived cells from skeletal muscle of C57Bl/6 animals. The resulting preparation is a mixture of many cell types including satellite cells (a.k.a., muscle progenitor cells) and hematopoietically active muscle-derived cells. - [Read Protocol for the Isolation of a Heterogenous Muscle-Derived Cell Population]

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

Quality is important in all aspects of tissue culture since the quality of materials used i.e. media and other reagents) will affect the quality of the cultures and products derived from them. The main areas of quality control that are of concern for tissue culture are: The quality of the reagents and materials; The provenance and integrity of the cell lines; The avoidance of microbial contamination. - [Read Quality Control Considerations for Cell Culture]

Category: PCR Protocols: cDNA Synthesis Protocols

This method is used to extend partial cDNA clones by amplifying the 5' sequences of the corresponding mRNAs.  The technique requires knowledge of a small region of sequence within the partial cDNA clone.  During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence. - [Read Rapid Amplification of 5' cDNA Ends 5'-RACE Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cytotoxicity Assays in Cell Culture Protocols

Rabbit-derived corneal cells are cultured in the presence of test compounds, the toxicity of which are determined by their effect upon cell viability.  A decrease in cell number, as measured by uptake of the dye Neutral Red, serves as an indicator of potential cytotoxicity.  This test has been proposed as a potential replacement alternative for the Draize Eye Irritation test. - [Read SIRC Cytotoxcitiy Test]

Category: Antibody Protocols: Antibody Handling Protocols

Ascitic fluid from B-cell-derived tumors is an extremely concentrated solution of specific antibodies. Ascitic fluid is often stored at -70ºC to prevent proteolytic degradation. - [Read Storage of Ascitic Fluid Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

A synthetic oligonucleotide annealed to single-stranded DNA derived from a recombinant bacteriophage M13 or phagemid template is used to prime the synthesis of complementary radiolabeled DNA. Synthesis is catalyzed by the Klenow fragment of E. coli DNA polymerase I, which extends the annealed primer for various distances along the single-stranded template DNA. - [Read Synthesis of Single-stranded DNA Probes of Defined Length from Bacteriophage M13 Templates Protocol]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Transfection of primary leukocytes has traditionally been a challenging but much desired protocol. It allows not only the analysis of cells in a more natural state to a cell line system, it enables the direct comparison of, for e.g. transcriptional activity using luciferase reporters, in immune cells taken from genetically-altered mice. In addition, importantly it allows for "rescue experiments" in knockout cells & the ability to over-express or reconstitute wild-type and/or mutated constructs. - [Read Transfection of Bone Marrow-Derived Mast Cells for Transcription Factor Luciferase Reporter Assays]