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1 10 100 1 10 100 |i-Galaetot[dase activity

Figure 6 Analysis of intracellular gene expression. A B-cell line was generated, as a model of differentiation, which upon stimulation expresses k light chain and turns off the expression of DNA elements controlling the artificially introduced bacterial lacZ gene. The histogram (top) demonstrates that /acZ (E. coli 13-galactosidase) expression is reduced following lipopolysaccha-ride (LPS) stimulation. The /3-galactosidase enzyme activity is measured by introducing a fluorogenic substrate, fluorescein digalactoside, into the cells. The contour plots demonstrate a concomitant increase in k light chain expression, as detected by surface immunofluorescence. (Courtesy of Dr William Kerr.)

1 10 100 1 10 100 |i-Galaetot[dase activity

Figure 6 Analysis of intracellular gene expression. A B-cell line was generated, as a model of differentiation, which upon stimulation expresses k light chain and turns off the expression of DNA elements controlling the artificially introduced bacterial lacZ gene. The histogram (top) demonstrates that /acZ (E. coli 13-galactosidase) expression is reduced following lipopolysaccha-ride (LPS) stimulation. The /3-galactosidase enzyme activity is measured by introducing a fluorogenic substrate, fluorescein digalactoside, into the cells. The contour plots demonstrate a concomitant increase in k light chain expression, as detected by surface immunofluorescence. (Courtesy of Dr William Kerr.)

performed. These studies are necessary for understanding the pathogenic consequences of changes in the representation of fine lymphocyte subsets.

Molecular and functional studies

More and more frequently, the FACS is used in a wide variety of immunologically relevant molecular studies. These range from measuring changes in the concentration of intracellular small molecules (physiological state or signal transduction events) to quantitating gene expression to chart activation or differentiation events.

In the late 1980s, the FACS molecular tool kit was extended by the development of a technique for measuring the commonly-used reporter gene lacZ (ยก3-galactosidase) within the intracellular compartment of mammalian (as well as bacterial and insect) cells. Depending on how it is introduced, this gene can be used cither as a marker for lineage and migration studies or as an insertion probe that can reveal differentiation-dependent enhancer and promoter sites (Figure 6, see p. 938). Furthermore, when introduced as a reporter gene under the control of particular promoter and enhancer elements, it can be used to investigate the mechanisms that regulate gene expression or provide a quantitative indicator of shifts in the activation or differentiation status of individual cells (Figure 7).

Recent developments using inherently fluorescent proteins have provided another tool in the arsenal of FACS-based reporter gene studies. The first of these proteins, termed green fluorescent protein (GFP), has spectral characteristics marginally suitable for quantitation by flow cytometry. An enormous effort has gone into the generation of mutants of the original GFP which have not only increased brightness, but different spectral properties as well. Currently, there are two versions of the GFP which can be simultaneously and independently quantitated. This brings

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