invitrogen Protocols

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

Incorporation of radioactive nucleotides to monitor ds cDNA synthesis. Invitrogen - [Read Incorporation of radioactive nucleotides to monitor ds cDNA synthesis]

Category: Immunology: T Cell Protocols

Describes generating CTL against some commonly used target antigens. Two methods for the quantitation of CTL activity are described based on the two pathways used bt CTL to kill target cells. In one pathway, they release lytic granules containing perforin and granzymes, leading to apoptosis and target cell lysis. In a second pathway, they trigger apoptosis via Fas/Fas ligand interactions. - [Read Induction and Measurement of Cytotoxic T Lymphocyte Activity Protocol]

Category: Immunology: T Cell Protocols

Describes methods for labeling high or low numbers of lymphocytes with CSFE. Protocols are provided to use CSFE-labeled cells in cell transfer studies or as cells to be cultured in vitro. Detailed guidelines for positioning of CSFE-labeled lymphocytes in lymphoid organs or other tissues are included for those wishing to use this approach to study lymphocyte migration. - [Read Intracellular Fluorescent Dye CFSE to Monitor Lymphocyte Migration and Proliferation]

Category: Immunology: Mononuclear Cell Isolation Protocols

Presents two methods for preparing dendritic cells (DCs), a highly specialized type of antigen-presenting cell (APC). The first method involves the isolation of DCs from mouse spleen, resulting in a cell population that is highly enriched in accessory cell and APC function. A support protocol for collagenase digestion of splenocyte suspensions is described to increase the yield of dendritic cells. The second method involves generating large numbers of DCs from mouse bone marrow progenitor cells. - [Read Isolation of Dendritic Cells Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: B Lymphocyte Isolation Protocols

A flow cytometry technique is presented, which results in the selection and isolation of two populations of cells from a complex mixture based on physical properties (e.g., size and internal granularity) and correlated expression of several surface molecules - [Read Isolation of Ly-1+/CD5+ B Cells by Cell Sorting Protocol]

Category: Mouse Protocols

Provides two protocols that may be used to isolate IHL. One can be used to isolate IHL from multiple livers in parallel, whereas the more elaborate alternate protocol yields more cells per liver but is more appropriately used to recover the IHL from a single liver. - [Read Isolation of Mouse Intrahepatic Lymphocytes Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols

Covers techniques for preparing lymphoid cell suspensions and removing red blood cells and dead cells. - [Read Isolation of Mouse Mononuclear Cells Protocol]

Category: Immunology: T Cell Protocols

Peyer’s Patch, and Lamina Propria Cells lymphocyte populations should be analyzed when studying the immunological status of the intestine, for example in oral immunization or in intestinal disease (including infectious disease and tumors). This protocol details techniques for isolation of IEL, PP cells, and LP cells from the small intestine of the mouse. - [Read Isolation of Mouse Small Intestinal Intraepithelial Lymphocytes 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: Cell Culture Protocols

Information on the methods used to cultivate large amounts of cells for the purpose of obtaining an endogenous or recombinant product. - [Read Large-Scale Cell Culture Protocol]

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

Measurement of Apoptosis and Other Forms of Cell Death. Jagan Muppidi, Melissa Porter, and Richard M. Siegel. As programmed cell death (PCD) or apoptosis has emerged as an important regulator of development and homeostasis in multicellular organisms, methods to quantify apoptosis and to distinguish it from necrosis have been developed. This unit presents a set of assays for these purposes, many of which are technically very simple and ideally suited to the study of hematopoietic cells. - [Read Measurement of Apoptosis and Other Forms of Cell Death]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

The more commonly available single-laser cytometers can also be used to measure multicellular conjugates, but due to overlaps in emission spectra, the extent of labeling cells with fluorophores must be controlled much more carefully when the single-laser machines are used. This protocol describes the labeling of cells and analysis of conjugates with either dual-laser or single laser flow cytometers. - [Read Measurement of Intercellular Conjugates by Flow Cytometry Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Intracellular Ion Measurement Protocols

Describes flow cytometric protocols using the dyes Indo-1 AM, Fluo-3, and Fura Red AM to measure intracellular calcium concentration. Support protocols detail the use of calcium buffers to calibrate a flow cytometric calcium assay, and methods to facilitate dye loading; an alternate protocol describes the use of a spectrofluorimeter to measure intracellular calcium for those investigators without access to a flow cytometer. - [Read Measurement of Intracellular Ions by Flow Cytometry Protocol]

Category: Yeast Cell Culture Protocols: Yeast Media, Solutions and Stocks Protocols

The yeast, Saccharyomyces cerevisiae, has become an important organism in molecular, biochemical, and genetic analysis. The organism has specific requirements for growth under a variety of conditions. The media, both liquid and solid, simple, define, and complex are describe in this unit. Also included are methods for handling, storing, and shipping stock of yeast. - [Read Media and Culture of Yeast Protocol]

Category: Cell Biology and Cell Culture: Cell Culture Growth Media: Mammalian Cell Culture Growth Media Protocols

The culture medium is an essential component of the in vitro environment and must be selected or designed with care. This protocol provides guidelines for design of serum-containing and serum-free media, selective and specialty media, and media for growth under special conditions such as soft-agar growth. - [Read Media for Culture of Mammalian Cells Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Cycle Analysis Protocols

Exponentially growing cells are asynchronous with respect to the cell cycle stage. Detection of cell cycle-related events is improved by enriching the culture for cells at the stage during which the particular event occurs. Methods for synchronizing cells are provided here, including those based on morphological features of the cell. - [Read Methods for Synchronizing Cells at Specific Stages of the Cell Cycle]

Category: PCR Protocols: PCR Cloning Protocols

The efficiency of direct cloning of PCR products can be improved by generating suitable ends on the amplified fragments.  This protocol describes the strategies for generating and manipulating suitable ends on the PCR fragments. - [Read Molecular Cloning of PCR Products Protocol]

Category: Molecular Imaging

Molecular Probes at Invitrogen - [Read Molecular Probes at Invitrogen]

Category: Molecular Imaging

The Handbook — A Guide to Fluorescent Probes and Labeling Technologies is a comprehensive resource for fluorescence technology and its applications. Newly revised, The Handbook contains detailed information describing the use of more than 3000 Molecular - [Read Molecular Probes Handbook Invitrogen]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Flow Cytometry Immunofluorescence Staining Protocols

Provides a brief historical perspective to illustrate the demands for this technology and to lay the foundation for its application; Explains the hurdles that were surmounted to achieve the current level of multiparametric analysis which serves to alert researchers to potential problems they may encounter when they either bring this technology to their own laboratories, or when they use extant instrumentation in another laboratory; Illustrates some of the complexities that arise. - [Read Multiparameter FACS Analysis]

Category: PCR Protocols: Real-Time PCR Protocols

In multiplex real-time PCR, different sets of primers with different labels are used to amplify separate genes from the template DNA in one tube.  This protocol uses LUX (Light Upon eXtension) primers from invitrogen.  FAM (6-carboxy-fluorescein) is used to label the gene of interest, and JOE (6-carboxy-4', 5'-dichloro-2',7'-dimethoxy-fluorescein) is used to label a housekeeping gene as an internal control to normalize between different reactions. - [Read Multiplex Real-Time PCR Protocol]

Category: Protein Protocols: Western Blot Protocol: Troubleshooting Western Blots

Optimizing Western Blots: Pre-Transfer Invitrogen - [Read Optimizing Western Blots: Pre-Transfer Invitrogen]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Provides an overview of terminology and echniques unique to flow ytometry and is divided into two sections. The first section discusses technical aspects of flow cytometry which apply primarily to the instrumentation-oriented flow cytometry phase. The second section discusses electronic cell separation using flow cytometry. - [Read Overview of Flow Cytometry]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

A procedure for direct and indirect staining of single-cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single-cell suspensions is also included. - [Read Preparation of Cells and Reagents for Flow Cytometry Protocols]

Category: Cell Biology and Cell Culture: Endothelial Cell Protocols

Endothelial cells, which line blood vessels, can be prepared from a variety of tissues. They are frequently prepared from the umbilical vein, which is relatively easy to obtain. The procedure is clearly described and provides a large population of highly purified endothelial cells. There are also methods for obtaining endothelial cells from other tissues such as fat, skin, and mucosa. These methods require special care and generate smaller populations of cells. - [Read Preparation of Endothelial Cells Protocol]