细胞功能测定

Traditionally, the study of human T cell development has relied on the availability of human and mouse thymic tissue. In this chapter, we outline a simple in vitro protocol for generating large numbers of human T-lineage cells from umbilical cord blood (CB)- derived hematopoietic stem cells (HS ...

The successful isolation and characterization of human embryonic stem cells (hESCs) provides a powerful tool to study the cellular and genetic mechanisms that mediate cell-fate decisions toward distinct developmental lineages. hESC-derived cells may also be suitable for novel c ...

In traditional bone marrow transplantation, the majority of peripherally introduced stem cells are trapped in peripheral organs, such as the lung and liver. The frequency of cells homed in bone marrow by such method is extremely low. This circumstance adds difficulty to the research of hemat ...

The “side population” (SP) phenotype is a manifestation of primitive cells’ ability to efficiently efflux the fluorescent DNA-staining dye Hoechst 33342 and can be used as the basis by which to isolate these cells using flow cytometry. In the bone marrow (BM), the SP defines a cell subset with a highly h ...

Complementing mutant embryos or embryonic stem cells with normal cells in embryonic chimeras is a valuable tool for investigating phenotypes. Chimera approaches provide a method to examine the phenotype of mutant cells, including hematopoiesis, in mutants with early embryonic le ...

Hematopoietic stem cell transplantation has traditionally been used to reconstitute blood cell lineages that had formed abnormally because of genetic mutations, or that had been eradicated to treat a disease such as leukemia. However, in recent years, much attention has been paid to the new ...

Hematopoietic stem cells (HSC) are inherently rare cell types that cannot be obtained in sufficient amounts for classical biochemical characterization. To facilitate functional studies of murine HSC and hematopoietic development, the technique of retroviral-mediated gene ...

The birth of viable offspring from somatic cell nuclear transfer (SCNT) in mammals caused a major re-examination of the understanding of the commitment of cells to specific tissue lineages during differentiation. The questions of whether cells undergo dedifferentiation or transd ...

This chapter describes a procedure for isolation and analysis of fractions enriched in plasma membranes from minute amounts of tissue. It consists of a method for extraction and fractionation of membranes and a method for enzymatic digestion of membrane proteins without use of detergen ...

Sequential detergent extraction of proteins from eukaryotic cells has been used to increase proteome coverage of 2D-PAGE. We have adapted sequential detergent extraction for use with the high-throughput non-electrophoretic proteomics method of liquid chromatography and el ...

Mitochondria are essential organelles in cellular metabolism. These organelles are bounded by two membranes, the outer and inner membrane. Especially the inner membrane comprises a high content of proteins, for example, the protein complexes of the respiratory chain. High-resolu ...

Identification and characterization of membrane proteins is of increasing importance in modern proteomic studies. It is of central interest to have access to methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. Separation me ...

Generally, a combination of two or more chromatographic and/or electrophoretic methods is conducted to separate membrane protein complexes. Here we describe how thylakoid membrane protein complexes from the photosynthetic apparatus can be successfully separated by two main s ...

Pancreatic zymogen granules (ZG) are specialized for digestive enzyme storage and regulated secretion in the exocrine pancreas and are a classical model for studying secretory granule function. To understand the function of this organelle, we have conducted a proteomic study to iden ...

Membrane proteomic analysis is of considerable interest due to the role of receptors, ion channels, and membrane-associated proteins that are critical components in cellular control and differentiation. Consequently, proteomic investigations of membrane proteins under a v ...

Liquid chromatography (LC) can be combined with matrix-assisted laser-desorption ionization (MALDI) mass-spectrometry (MS) by using automated off-line fraction collection of eluates onto a MALDI plate. Commercial tandem MS systems are available for generating product ion sp ...

Although two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used as the standard proteomic approach for separating proteins in a complex mixture, this technique has many drawbacks. These include a limited molecular mass range, poor separation of highly acidic ...

G-protein coupled receptors (GPCRs) are a superfamily of membrane integral proteins responsible for a large number of physiological functions. Approximately 50% of marketed drugs are targeted toward a GPCR. Despite showing a high degree of structural homology, there is a large variance ...

The nuclear envelope (NE) is a double membrane system that is both a part of the endoplasmic reticulum and part of the nucleus. As its constituent proteins tend to be highly complexed with nuclear and cytoplasmic components, it is notoriously difficult to purify. Two methods can reduce this diffic ...

Lipid rafts are small subdomains of the plasma membrane enriched in cholesterol, sphingolipids, saturated phospholipids and specific proteins. They are thought to act as coordination centres for signal transduction pathways so their protein composition is of particular biolo ...