Cytogenetic analysis forms an essential part of characterizing and identifying cell lines, in particular those established from tumors. In addition, karyotypic analysis can be used to distinguish individual subclones and to monitor stability. This chapter describes basic cyto ...

Hematopoietic stem cells present in small numbers in certain fetal organs during development and in adult bone marrow produce a heterogeneous pool of progenitors that can be detected in vitro using colony-forming cell (CFC) assays. Hematopoietic progenitor cells, when cultured in a sem ...

The two most convenient sources of primary murine macrophages are the bone marrow and the peritoneal cavity. Resident peritoneal macrophages can readily be harvested from mice and purified by adherence to tissue culture plastic. The injection of Bio-Gel polyacrylamide beads or thiog ...

Methods to isolate and culture human monocyte-derived macrophages and alveolar macrophages are described. Monocytes are obtained from buffy-coat preparations by Ficoll density gradient centrifugation, followed by adhesion-mediated purification on tissue culture or g ...

Fetal thymus organ culture (FTOC) is a unique and powerful culture system that allows intrathymic T-lymphocyte development in vitro. T-cell development in FTOC well represents fetal thymocyte development in vivo. Here, we describe the basic method for FTOC as well as several related techn ...

The marriage of cell and molecular biology with embryology has produced remarkable advances for the field of developmental biology. In this third volume of Developmental Biology Protocols, contemporary, practical methods are first presented for the analysis and manipulation of d ...

Ectopic expression in Drosophila has been used extensively to examine the capabilities of a given gene in virtually any tissue. Three general approaches are described here, and the choice of which to use is determined by the needs of the particular experiment. Certain aspects of each approach c ...

Clonal analysis in Drosophila has been successfully used to address numerous biological questions of fundamental importance, including issues of cell lineage, fate determination, autonomy of gene action and pattern formation (1, 2). Clonal analysis has been particularly useful f ...

The current trend in developmental biology research is to identify candidate functional genes and then manipulate their expression or activity by either gain or loss of function to elucidate the specific roles of their protein products. One such procedure is the introduction of antigen- ...

Perturbing the expression level of a specific gene in vivo provides a powerful approach towards explaining its function during embryonic development. One technique used for perturbing the level of expression of a specific gene is the application of gene-specific antisense oligodeo ...

in situ hybridization has become a powerful tool for the analysis of gene expression within a topographical context and has become indispensable in developmental studies. Different strategies are to be used for different purposes. Whole mount in situ hybridization allows the rapid glo ...

Over the last several decades, an understanding of the events involved in embryonic pattern formation has been extended from an anatomic to a molecular level. In particular, the ability to examine the spatial and temporal expression of specific genes has aided greatly the analysis of their fu ...

Two methods are commonly employed to characterize the spatiotemporal aspect of gene expression: 1. in situ hybridization (ISH), which localizes mRNA transcripts using a labeled nucleotide probe and 2. immunohistochemistry (IMH), which detects protein gene products by means of labe ...

Whole mount in situ hybridization (WMISH) is a method for detecting specific messenger ribonucleic acids (mRNAs) at their site of expression within an embryo or intact tissue fragment. Like conventional in situ hybridization, WMISH depends upon the availability of a complementary nuc ...

In situ hybridization is a procedure that allows the detection of the site(s) of transcription of a given gene at a cellular level within an entire organism. Since its introduction into developmental biology (1), this procedure has become an indispensable tool to investigate gene expression; ...

The distribution of two different molecules can be analyzed within the same embryo using the procedures described below. The protocol for combined whole mount in situ hybridization and immunocytochemistry allows for simultaneous detection of messenger ribonucleic acid (mRNA) a ...

One of the most remarkable and unexpected advances in cell biology in the 1990s has been the introduction of expression plasmids containing a wide variety of cDNAs ligated to the cDNA of the protein responsible for the bioluminescence of the jellyfish, Aequoria victoria (1, 2). The original expr ...

Determination and differentiation of tissues during embryogenesis of multicellular organisms relies on differential utilization of genetic information in different cell types at different stages of development, resulting in unique patterns of gene expression that impa ...

The ribonuclease (RNase) protection assay provides a highly sensitive method for the detection and quantitation of specific RNAs from tissues and cells as well as for the analysis of mRNA and gene structure (1). This solution hybridization approach is at least 10-fold more sensitive than Nor ...

Quantitation of very small amounts of specific messenger ribonucleic acids (mRNAs) extracted from tissues or cell cultures is a difficult and time-consuming task that necessitates the use of carefully controlled amplification procedures. Reverse transcription (RT) of the mRNA to ...