Rapid flow cytometric method for measuring senescence associated beta-galactosidase activity in human fibroblasts.
AuthorsNoppe G, Dekker P, de Koning-Treurniet C, Blom J, van Heemst D, Dirks RW, Tanke HJ, Westendorp RG, Maier AB,
JournalCytometry A
PubMed ID19777541
'Senescence associated-beta-galactosidase (SA-beta-gal) activity is a widely used marker for cellular senenescence. SA-beta-gal activity is routinely detected cytochemically, manually discriminating negative from positive cells. This method is time-consuming, subjective and therefore prone to operator-error. We aimed to optimize a flow cytometric method described by other workers using endothelial cells to ... More
Optical imaging fiber-based single live cell arrays: a high-density cell assay platform.
AuthorsBiran I, Walt DR
JournalAnal Chem
PubMed ID12141663
'A high-density, ordered array containing thousands of microwells is fabricated on an optical imaging fiber. Each individually addressable microwell is used to accommodate a single living cell. A charged coupled device (CCD) detector is employed to monitor and spatially resolve the fluorescence signals obtained from each individual cell, allowing simultaneous ... More
An optimized electroporation protocol applicable to a wide range of cell lines.
AuthorsBaum C, Forster P, Hegewisch-Becker S, Harbers K
JournalBiotechniques
PubMed ID7873174
'A number of transfection methods for mammalian cells are available; however, many cell lines may appear resistant to efficient transfection, or at best, necessitate lengthy optimization procedures in recommended protocols. We describe here an electroporation protocol that yields highly efficient gene transfer (20%-100% of surviving cells) in all 19 cell ... More
Mapping mechanisms and charting the time course of premature cell senescence and apoptosis: lysosomal dysfunction and ganglioside accumulation in endothelial cells.
AuthorsPatschan S, Chen J, Gealekman O, Krupincza K, Wang M, Shu L, Shayman JA, Goligorsky MS,
JournalAm J Physiol Renal Physiol
PubMed ID17928415
'Endothelial cells subjected to glycated collagen I develop premature senescence within 3-5 days, as revealed by increased senescence-associated beta-galactosidase activity, decreased proliferation, and an increase in cell size. Here, we analyzed the time course and possible mechanisms of this process. Lysosomal integrity studies revealed a rapid collapse of pH gradient ... More
Use of fluorescence-activated cell sorting for rapid isolation of insect cells harboring recombinant baculovirus.
AuthorsSommerfelt MA, Sorscher EJ
JournalMethods Cell Biol
PubMed ID7877509
Detecting lacZ gene expression in living cells with new lipophilic, fluorogenic beta-galactosidase substrates.
AuthorsZhang YZ, Naleway JJ, Larison KD, Huang ZJ, Haugland RP
JournalFASEB J
PubMed ID1720751
'Current methods for detecting lacZ expression in transformed cells are limited because they require such harsh conditions that viability of the cells after detection is drastically reduced. To overcome this problem, we developed a series of new substrates for detection of lacZ expression in living cells under standard culture or ... More
Detecting enzymatic activity in cells using fluorogenic substrates.
AuthorsHaugland RP
JournalBiotech Histochem
PubMed ID8580208
Fluorogenic substrates can detect enzymatic activity associated with cells. It is difficult, however, to detect activity within a single cell or in an organelle since hydrolytic substrates yield products that rapidly leak from the cell. Several new solutions are presented including trapping the fluorescent product in membranes, in cell organelles, ... More
Use of fluorescein-di-beta-D-galactopyranoside (FDG) and C12-FDG as substrates for beta-galactosidase detection by flow cytometry in animal, bacterial, and yeast cells.
AuthorsPlovins A, Alvarez AM, Ibañez M, Molina M, Nombela C
JournalAppl Environ Microbiol
PubMed ID7811104
Fluorescein-di-beta-D-galactopyranoside (FDG) was found to be a useful substrate for beta-galactosidase detection by flow cytometry in gram-negative bacteria, since it entered viable cells and gave a fluorescence emission proportional to the enzymatic activity. C12-FDG, a more lipophilic derivative, gave a very poor signal because of the lack of penetration. On ... More
Rapid purification of recombinant baculovirus using fluorescence-activated cell sorting.
Authors Peng S; Sommerfelt M A; Berta G; Berry A K; Kirk K L; Hunter E; Sorscher E J;
JournalBiotechniques
PubMed ID8431294
Expression of foreign proteins in the baculovirus-insect cell expression system has been limited by difficulties in rapid identification and purification of recombinant virus. Although the process of identifying recombinant virus has been greatly facilitated by the introduction of vectors that lead to insect cell co-expression of beta-galactosidase with foreign genes ... More
Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo.
AuthorsDebacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O,
JournalNat Protoc
PubMed ID20010931
Normal cells can permanently lose the ability to proliferate when challenged by potentially oncogenic stress, a process termed cellular senescence. Senescence-associated beta-galactosidase (SA-betagal) activity, detectable at pH 6.0, permits the identification of senescent cells in culture and mammalian tissues. Here we describe first a cytochemical protocol suitable for the histochemical ... More
Functional protein delivery into neurons using polymeric nanoparticles.
AuthorsHasadsri L, Kreuter J, Hattori H, Iwasaki T, George JM,
JournalJ Biol Chem
PubMed ID19129199
An efficient route for delivering specific proteins and peptides into neurons could greatly accelerate the development of therapies for various diseases, especially those involving intracellular defects such as Parkinson disease. Here we report the novel use of polybutylcyanoacrylate nanoparticles for delivery of intact, functional proteins into neurons and neuronal cell ... More
Nucleic acid detection using non-radioactive labelling methods.
AuthorsMansfield ES, Worley JM, McKenzie SE, Surrey S, Rappaport E, Fortina P
JournalMol Cell Probes
PubMed ID7477006
Nucleic acid probe-based assays are now widely used in genetic research, human identification, forensics and in a broad spectrum of clinical assays in the fields of microbiology, haematology/oncology and virology. Labelled probes are used in a variety of assay formats including dot-blots, Southern blots (DNA target), Northern blots (RNA target), ... More
Verapamil inhibition of enzymatic product efflux leads to improved detection of beta-galactosidase activity in lacZ-transfected cells.
AuthorsPoot M, Arttamangkul S
JournalCytometry
PubMed ID9136753
The beta-galactosidase activity encoded by the lacZ gene of Escherichia coli is widely used to monitor successful expression of transfected genes. Fluorogenic substrates allow detection of enzyme activity in viable cells, which, subsequently, can be selected for further study on the basis of fluorescence emission. We analyzed three fluorogenic substrates ... More
Enzyme cytochemical techniques for metabolic mapping in living cells, with special reference to proteolysis.
AuthorsBoonacker E, Van Noorden CJ
JournalJ Histochem Cytochem
PubMed ID11724895
Specific enzymes play key roles in many pathophysiological processes and therefore are targets for therapeutic strategies. The activity of most enzymes is largely determined by many factors at the post-translational level. Therefore, it is essential to study the activity of target enzymes in living cells and tissues in a quantitative ... More
Specific activation of mammalian Hox promoters in mosaic transgenic zebrafish.
AuthorsWesterfield M, Wegner J, Jegalian BG, DeRobertis EM, Püschel AW
JournalGenes Dev
PubMed ID1348485
Homeo box-containing genes (Hox) are expressed in restricted regions of vertebrate embryos and may specify positional information. The organization and expression patterns of these genes are highly conserved among different species, suggesting that their regulation may also have been conserved. We developed a transient expression system, using mosaically transgenic zebrafish, ... More
Analysis of Escherichia coli beta-galactosidase expression in transgenic mice by flow cytometry of sperm.
AuthorsJasin M, Zalamea P
JournalProc Natl Acad Sci U S A
PubMed ID1438265
Alterations to the mammalian genome that occur during the development of germ cells, in particular during meiosis, can be introduced into the population upon fertilization. These alterations can occur through homologous recombination, genome rearrangement, or mutagenesis. Such events usually occur infrequently for any particular sequence. Because of the difficulty in ... More
Measurement of hydrolysis kinetics of galactose-substituted fluorescein by beta-galactosidase at the toluene-water interface by spinning microtube fluorometry.
AuthorsOkamoto Y, Monjushiro H, Fukumoto T, Watarai H
JournalAnal Bioanal Chem
PubMed ID16838159
A new analytical technique, spinning microtube fluorometry (SMF), was developed and applied to the study of interfacial hydrolysis of 5-dodecanoylaminofluorescein di-beta-D: -galactopyranoside (C12FDG) by beta-galactosidase (beta-gal) in the toluene-water system. The nonfluorescent lactone form of C12FDG in the toluene phase was converted at the interface to 5-dodecanoylaminofluorescein (C12F), which was ... More
Effect of amplification of dhfr and lac Z genes on growth and beta-galactosidase expression in suspension cultures of recombinant CHO cells.
AuthorsGu MB, Kern JA, Todd P, Kompala DS
JournalCytotechnology
PubMed ID1369176
Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the gene for human beta-interferon (beta-IFN) or the lac Z gene which ... More
Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells.
AuthorsKurz DJ, Decary S, Hong Y, Erusalimsky JD
JournalJ Cell Sci
PubMed ID11017877
Senescence-associated (beta)-galactosidase is widely used as a biomarker of replicative senescence. However, it remains unknown whether this is a distinct enzyme active at pH 6, and differentially expressed in senescence, or a manifestation of an increase in the classic acid lysosomal (beta)-galactosidase. Here we have investigated the origin of senescence-associated-(beta)-galactosidase ... More
Effect on heterocyst differentiation of nitrogen fixation in vegetative cells of the cyanobacterium Anabaena variabilis ATCC 29413.
AuthorsThiel T, Pratte B
JournalJ Bacteriol
PubMed ID11114927
Heterocysts are terminally differentiated cells of some filamentous cyanobacteria that fix nitrogen for the entire filament under oxic growth conditions. Anabaena variabilis ATCC 29413 is unusual in that it has two Mo-dependent nitrogenases; one, called Nif1, functions in heterocysts, while the second, Nif2, functions under anoxic conditions in vegetative cells. ... 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
Targeted gene delivery to alveolar macrophages via Fc receptor-mediated endocytosis.
AuthorsRojanasakul Y, Wang LY, Malanga CJ, Ma JK, Liaw J
JournalPharm Res
PubMed ID7899236
Alveolar macrophage (AM) plays important roles in lung homeostasis and pathogenesis of diseases. The study of macrophage gene function and regulation as well as its potential therapeutic intervention will require the development of vectors capable of safe and efficient transfer of DNA to the AM. In the present study, we ... More
Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses.
AuthorsDavey HM, Kell DB
JournalMicrobiol Rev
PubMed ID8987359
The most fundamental questions such as whether a cell is alive, in the sense of being able to divide or to form a colony, may sometimes be very hard to answer, since even axenic microbial cultures are extremely heterogeneous. Analyses that seek to correlate such things as viability, which is ... More
Transmembrane topology of alpha- and beta-subunits of Na+,K+-ATPase derived from beta-galactosidase fusion proteins expressed in yeast.
AuthorsFiedler B, Scheiner-Bobis G
JournalJ Biol Chem
PubMed ID8910592
Various models of the transmembrane topology of the Na+,K+-ATPase predict either 8 or 10 membrane spans for the alpha-subunit and one to three membrane spans for the beta-subunit. Structure/function analysis, however, requires precise knowledge about the folding of enzymes. Therefore, the intention of this work was to establish a transmembrane ... More
Using proteolysis-targeting chimera technology to reduce navitoclax platelet toxicity and improve its senolytic activity.
Authors
JournalNat Commun
PubMed ID32332723
Senescence-associated reprogramming promotes cancer stemness.
Authors
JournalNature
PubMed ID29258294
Overexpression of COMP-Angiopoietin-1 in K14-Expressing Cells Impairs Hematopoiesis and Disturbs Erythrocyte Maturation.
Authors
JournalMol Cells
PubMed ID33935045
Fluorescence in situ Localization of Gene Expression Using a lacZ Reporter in the Heterocyst-forming Cyanobacterium Anabaena variabilis.
Authors
JournalBio Protoc
PubMed ID34458415
Deletion of SA β-Gal+ cells using senolytics improves muscle regeneration in old mice.
Authors
JournalAging Cell
PubMed ID34904366
Autophagy drives fibroblast senescence through MTORC2 regulation.
Authors
JournalAutophagy
PubMed ID31931659
Aberrant Induction of a Mesenchymal/Stem Cell Program Engages Senescence in Normal Mammary Epithelial Cells.
Authors
JournalMol Cancer Res
PubMed ID33443106
A non-genetic, cell cycle-dependent mechanism of platinum resistance in lung adenocarcinoma.