'Mouse embryonic stem (ES) cells were transfected with a plasmid composed of an E. coli lacZ gene fused to 1.8 kb of rat neuron-specific enolase (NSE) promoter sequences. While this reporter construct had been shown previously to function exclusively in postmitotic neurons and neuro-endocrine cells of transgenic mice, stably transfected ... More
'Many areas of research today are based on enzymatic assays most of which are still performed as enzyme-linked immunosorbent assays in microtiter plates. The demand for highly parallel screening of thousands of samples eventually led to a miniaturization and automation of these assays. However, the final transfer of enzymatic assays ... More
Microchip device for performing enzyme assays.
AuthorsHadd AG, Raymond DE, Halliwell JW, Jacobson SC, Ramsey JM
JournalAnal Chem
PubMed ID9286159
'An automated enzyme assay was performed within a microfabricated channel network. Precise concentrations of substrate, enzyme, and inhibitor were mixed in nanoliter volumes using electrokinetic flow. Reagent dilution and mixing were controlled by regulating the applied potential at the terminus of each channel, using voltages derived from an equivalent circuit ... More
Binding of alkyl 1-thio-beta-D-galactopyranosides to beta-D-galactosidase from E. coli.
AuthorsDe Bruyne CK, Yde M
JournalCarbohydr Res
PubMed ID18283
'The binding of a series of alkyl 1-thio-beta-D-galactopyranosides to beta-D-galactosidase from E. coli has been investigated. The inhibition constants were compared to the partition coefficients for the transfer of these substrate-analogues from water to 1-octanol. The relationships between the observed binding-constants and the partition coefficients indicate that part of the ... More
AuthorsLorincz M, Roederer M, Diwu Z, Herzenberg LA, Nolan GP
JournalCytometry
PubMed ID8866216
'We report the development of a new fluorescence-activated cell sorter (FACS)-based reporter gene system utilizing the enzymatic activity of the E. coli beta-glucuronidase (gus) gene. When loaded with the Gus substrate fluorescein-di-beta-D-glucuronide (FDGlcu), individual mammalian cells expressing and translating gus mRNA liberate sufficient levels of intracellular fluorescein for quantitative analysis ... More
Beta-galactosidase activity in single differentiating bacterial cells.
AuthorsRusso-Marie F, Roederer M, Sager B, Herzenberg LA, Kaiser D
JournalProc Natl Acad Sci U S A
PubMed ID8396263
'Myxococcus xanthus strains containing transcriptional fusions to lacZ were analyzed and fractionated by differences in their levels of beta-galactosidase expression. The fluorogenic substrate for beta-galactosidase, fluorescein di-beta-galactopyranoside, was introduced into M. xanthus cells during a rapid decrease in osmolarity of the medium followed by a return to isoosmolarity. Fluorescein, the ... More
Improved FACS-Gal: flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs.
AuthorsFiering SN, Roederer M, Nolan GP, Micklem DR, Parks DR, Herzenberg LA
JournalCytometry
PubMed ID1905992
'The previously reported FACS-Gal assay (Nolan et al., Proc Natl Acad Sci USA 85:2603-2607, 1988) measures E. coli lacZ-encoded beta-galactosidase activity in individual viable eukaryotic cells for a variety of molecular and cellular biological applications. Enzyme activity is measured by flow cytometry, using a fluorogenic substrate, which is hydrolyzed and ... More
A chemiluminescent assay for quantitation of beta-galactosidase in the femtogram range: application to quantitation of beta-galactosidase in lacZ-transfected cells.
AuthorsJain VK, Magrath IT
JournalAnal Biochem
PubMed ID1807155
An optimized chemiluminescent assay for beta-galactosidase using a chemiluminescent substrate AMPGD (3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1. 1(3,7)]decan]-4- yl)phenyl-beta-D-galactopyranoside) is described. This assay is rapid and sensitive and can detect as little as 2 fg of beta-galactosidase. Its use for the quantitation of beta-galactosidase in cells transfected with lacZ-expressing vectors is described. It is possible ... More
Acid beta-galactosidase: a developmentally regulated marker of endocrine cell precursors in the human fetal pancreas.
Isolation of endocrine cell precursors from the human fetal pancreas will be important to the study of islet cytodifferentiation and eventually for islet transplantation in insulin-dependent diabetes. These precursor cells, from which all four islet endocrine cell types arise, are present within fetal pancreatic ductal epithelium. After enzymatic digestion and ... More
A novel fluorescence-based system for assaying and separating live cells according to VDJ recombinase activity.
AuthorsYancopoulos GD, Nolan GP, Pollock R, Prockop S, Li SC, Herzenberg LA, Alt FW
JournalMol Cell Biol
PubMed ID2320007
We describe two retroviral vector-based recombination substrate systems designed to assay for lymphoid VDJ recombinase activity in cultured cells. Both substrates incorporate a constitutive dominant marker gene (the simian virus promoter-driven neo gene) to allow selection of cells that stably integrate the substrate. Both substrates also include a second marker ... More