invitrogen Protocols

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols: RNA Isolation fromTissues or Cultured Cells Protocols

The method uses Trizol, a phenol-base reagent produced by Invitrogen. - [Read Preparation of RNA from Paraffin-Embedded Fixed Tissue Using Trizol Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: T-Cell Isolation Protocols

Protocols utilize T hybridomas to detect expression of peptide-MHC complexes, since these cells provide the most convenient, consistent, and flexible T cell readout systems for these purposes. If desired, antigen-specific T cell clones can be used in lieu of T hybridoma cells, but T cell clones often give poorer responses than T hybridomas to fixed APCs due to fixation-induced loss of costimulator function. - [Read Presenting Exogenous Antigen to T Cells Protocol]

Category: Immunology: T Cell Protocols

T cell hybridomas can be obtained by fusing activated T cells with tumor cells. A heterogeneous population of hybridomas can be cloned by limiting dilution to obtain hybridomas that express specificity to one T cell receptor (TCR). Protocol describes cell fusion and selection of T cell hybridomas. A protocol is provided for screening of T cell hybridomas for expression of the CD3-TCR complex by flow cytometry analysis. - [Read Production of Mouse T Cell Hybridomas Protocol]

Category: Immunology: T Cell Protocols

Provides methods for the derivation of specific types of T cell clones: preparation and maintenance of alloreactive murine helper T (TH) lymphocyte and cytotoxic T lymphocyte (CTL) clones using the limiting dilution technique and derivation of TH clones reactive with soluble protein antigens including a method for the selection of either TH1 or TH2 lymphocyte subsets. - [Read Production of T Cell Clones Protocol]

Category: Immunology: B Cell Protocols

Describes procedures for measuring the capacity of purified B cells to undergo proliferation. The method centers on the use of polyclonal stimulating agents (mitogens) because these agents stimulate the majority of B cells and because the alternative (measurement of antigen-induced proliferation) requires the laborious procedures of isolating antigen-specific B cells (which are otherwise present in too low a concentration in whole B cell populations). - [Read Proliferative Assays for B Cell Function Protocol]

Category: Immunology: T Cell Protocols

Protocols describe the conditions required to induce proliferation are described. Also describe the assay in which CD4·CD25·T cells are co-cultured with conventional T cells in order to assess their suppressive function. Will describe the culture conditions for the activation and expansion of CD4·CD25· cells. - [Read Proliferative Assays for T Cell Function Protocols]

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols: RNA Isolation fromTissues or Cultured Cells Protocols

Protocol for purification of RNA from animal cells using Trizol. Trizol is a phenol-based reagent produced by Invitrogen. - [Read Purification of RNA from Animal Cells Using Trizol]

Category: Immunology: T Cell Protocols

In the first protocol, IL-2-producing murine T cells are measured following stimulation by the mitogen Con A. The second protocol provides a modification for using human responder cells. The second protocol is used for estimating the proportion of cells that can generate a clone of cytotoxic effector cells when stimulated by Con A with the addition of IL-2. - [Read Quantitation of Functional T Cells by Limiting Dilution Protocols]

Category: PCR Protocols: Quantitative PCR Protocols

Protocol that uses the polymerase chain reaction (PCR) to quantitate the numbers of a particular DNA sequence, from 1 to 20,000 molecules per sample.  In addition, it helps assess the presence of contaminating sequences, which can seriously affect the outcome of the procedure. - [Read Quantitation of Rare DNAs by PCR Protocol]

Category: PCR Protocols: Real-Time PCR Protocols

Protocol uses the Superscript II First-Strand Synthesis system for the generation of cDNA from total RNA.  RNA purified using TRIzol reagent (Invitrogen) or the methods described in Preparation of RNA Using Guanidinium Isothiocyanate/Cesium Chloride Ultracentrifugation, Preparation of RNA from Paraffin-Embedded Fixed Tissue. - [Read Real-Time RT-PCR: cDNA Synthesis Protocol]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol uses the Superscript II First-Strand Synthesis system for the generation of cDNA from total RNA.  RNA purified using TRIzol reagent (Invitrogen) or the methods described in Preparation of RNA Using Guanidinium Isothiocyanate/Cesium Chloride Ultracentrifugation, Preparation of RNA from Paraffin-Embedded Fixed Tissue. - [Read Real-Time RT-PCR: cDNA Synthesis Protocol]

Category: RNA Protocols: RT-PCR and cDNA synthesis: RT-PCR and cDNA Synthesis FAQ

RNase inhibitors and RNases - [Read RNase inhibitors and RNases]

Category: Cell Culture Protocols: Sterile Technique Protocols

This unit on sterility in the tissue culture environment describes methods for sterilization of liquid and dry goods used for tissue culture and filtration of liquids to prevent contamination of cultures. - [Read Sterilization and Filtration Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: T-Cell Isolation Protocols

Protocol describes removal of T cell subsets by cytotoxic elimination using CD-specific antibodies. Describes the complete removal of T cells from lymphocyte preparations based on the presence of the glycoprotein Thy-1 on the cell surface of T lymphocytes. Cytotoxic elimination is employed; however, Thy-1-specific antibodies are used rather than MHC class II-specific antibodies so that T cells are eliminated rather than B cells and accessory cells. - [Read T Cell Depletion by Cytotoxic Elimination Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: T-Cell Isolation Protocols

Describes T cell enrichment using cytotoxic antibodies, and also describes the depletion of T cells and their subpopulations using the same approach. In the latter unit, T cell surface markers (Thy-1, CD4, and CD8) are targeted by the cytotoxic antibodies. - [Read T Cell Enrichment by Cytotoxic Elimination of B Cells and Accessory Cells Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: T-Cell Isolation Protocols

Protocol describes a convenient, although imprecise, means of enriching T cells through removal of accessory and B cells; use of nylon wool is preferred if both of the latter subsets are to be removed, while Sephadex is used when the goal is primarily to remove accessory cells. - [Read T Cell Enrichment By Nonadherence to Nylon Protocol]

Category: DNA Protocols: TOPO Cloning

TOPO invitrogen Protocol - [Read TOPO invitrogen Protocol]

Category: RNA Protocols: RT-PCR and cDNA synthesis: RT-PCR and cDNA Synthesis FAQ

What is the highest temperature that a reverse trascriptase can be used? Invitrogen - [Read What is the highest temperature that a reverse trascriptase can be used?]

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast RNA Isolation Protocols

Protocol describes rapid, small-scale yeast RNA isolation. It is based on the work of Schmitt et al. (1990). Note that all containers should be washed in Rnase Away (Invitrogen) or dry baked for 24 hours at 160°C. - [Read Yeast RNA Isolation: Small-Scale Protocol]

Category: DNA Protocols: TOPO Cloning

Zero Blunt TOPO PCR Cloning Kit, Detailed protocol. (Invitrogen Life technologies). Alam Lab. Department of Microbiology, University of Hawai'i. - [Read Zero Blunt TOPO PCR Cloning Kit Protocol]