细胞生物学

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Generating Induced Pluripotent Stem Cells in Mouse Embryonic Fibroblast Cells

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors. Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that expr

Saturday,Jul 18,2009 Source: Jaenisch Lab, Whitehead Instit

Thomson Lab Stem Cell Protocols

A collection of stem cell protocols from Thomson lab including: Human and mouse embryonic stem cell Protocols and iPS cell protocols.

Saturday,Jul 18,2009 Source: Thomson Lab, University of Wis

HUMAN EMBRYONIC STEM CELL PROTOCOLS

A collection of stem cell culture protocols including the followings: General Notes on hESC Culture, Isolation of Primary Mouse Embryo Fibroblasts ,Thawing and preparing p1 MEF feeder plates, Preparation of MEF- Conditioned Medium (MEF-CM), Microdissection Passaging of hESCs, Bulk passaging of hESC , Cryopreservation of hESCs, Thawing of hESCs, Karyotyping, Media Recipes and Solutions, Tissue Culture Flasks and Plates, Reagents and suppliers.

Saturday,Jul 18,2009 Source: NIH Stem Cell Information

Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis

Flow cytometry is a powerful analytical technique in which individual cells can be simultaneously analyzed for several parameters, including size and granularity, as well as the expression of surface and intracellular markers defined by fluorescent antibodies

Saturday,Jul 18,2009 Source: BD Pharmingen

Immunofluorescence Staining and Flow Cytometry of Intracellular Cytokines

A modification of the basic immunofluorescence staining and flow cytometric analysis protocol can be used for the simultaneous analysis of surface molecules and intracellular cytokines at single-cell level. In this protocol, cells are first activated in vitro, stained for surface antigens, as in the regular staining protocol, then fixed and permeabilized to allow for anti-cytokine antibodies to stain intracellularly.

Saturday,Jul 18,2009 Source: eBioscience

Cytokine Expression Profiling

ELISPOT (Enzyme-linked ImmunoSPOT) technique was originally used to enumerate antibody secreting B cells. In the current technique, cells are deposited onto a membrane coated with one antibody specific for a protein followed by an appropriate incubation period. Subsequently, the protein of interest is detected in the environment immediately surrounding the cell secreting it, with another antibody specific for a different epitope of the protein. The signal detected by the HRP e

Saturday,Jul 18,2009 Source: eBioscience

Cytokine Elisa

Protocol for general use: Cytokine sandwich ELISA are sensitive enzyme immunoassays that can specifically detect and quantitate the concentration of soluble cytokine and chemokine proteins. The basic cytokine sandwich ELISA method makes use of highly-purified anti-cytokine antibodies (capture antibodies) which are noncovalently adsorbed (“coated” – primarily as a result of hydrophobic interactions) onto plastic microwell plates...

Saturday,Jul 18,2009 Source: BD Pharmingen

Cytokine Bioassays Using Neutralizing mAbs

Antibodies that block binding of cytokines to their specific receptors and neutralize their effects are critical in studies of cytokine function. The following three neutralization protocols describe in vitro bioassays using anti-mouse and anti-human cytokine antibodies. In general, the cytokine bioassay protocols are modified to pre-incubate the cytokine of interest with the specific neutralizing antibody prior to addition to the responding cells.

Saturday,Jul 18,2009 Source: eBioscience

Cytokine Bioassays

Biological activity of cytokines and their concentrations are commonly measured by cellular proliferation of primary cell cultures or isolated cell lines that are dependent and/or responsive to a specific growth factor. Other aspects of biological activity of cytokines include induction of further cytokine secretion, induction of killing, antiviral activity, degranulation, cytotoxicity, chemotaxis, and promotion of colony formation. In vitro assays to measure all of these acti

Saturday,Jul 18,2009 Source: eBioscience

The Under-Agarose Migration Assay

The Under-Agarose assay is a useful method for observing the response of a cell population to one or more chemoattractant sources. The behavior of individual migrating cells can be studied by modifying the assay for video microscopy.

Saturday,Jul 18,2009 Source: Bio.com

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