Qualification of embryonal carcinoma 2102Ep as a reference for human embryonic stem cell research.
AuthorsJosephson R, Ording CJ, Liu Y, Shin S, Lakshmipathy U, Toumadje A, Love B, Chesnut JD, Andrews PW, Rao MS, Auerbach JM
JournalStem Cells
PubMed ID17284651
'As the number of human embryonic stem cell (hESC) lines increases, so does the need for systematic evaluation of each line''s characteristics and potential. Comparisons between lines are complicated by variations in culture conditions, feeders, spontaneous differentiation, and the absence of standardized assays. These difficulties, combined with the inability of ... More
Quantitative differences in phase I and II metabolism between rat precision-cut liver slices and isolated hepatocytes.
'Testosterone (250 microM), 7-ethoxycoumarin (25 microM), and 1-chloro-2,4-dinitrobenzene (CDNB, 50 microM) were used as substrates to compare phase I and II metabolism in rat precision-cut liver slices and rat hepatocytes. Overall clearance to metabolites was significantly greater in hepatocytes for testosterone (1.9- to 16.9-fold), 7-ethoxycoumarin (O-deethylation, 14.8-fold; glucuronidation, 3.1-fold), and ... More
Alkaline phosphatase is involved in the control of adipogenesis in the murine preadipocyte cell line, 3T3-L1.
AuthorsAli AT, Penny CB, Paiker JE, van Niekerk C, Smit A, Ferris WF, Crowther NJ
JournalClin Chim Acta
PubMed ID15748605
'OBJECTIVE: As alkaline phosphatase may play a role in cell differentiation, our aim was to study the possible role of this enzyme in the differentiation of preadipocytes (3T3-L1 cells) into adipocytes. RESEARCH METHODS AND PROCEDURES: 3T3-L1 cells were grown in medium containing insulin, dexamethasone and IBMX to induce adipogenesis. Adipogenesis ... More
Fluorescence in situ hybridization of scarce leptin receptor mRNA using the enzyme-labeled fluorescent substrate method and tyramide signal amplification.
AuthorsBreininger JF, Baskin DG
JournalJ Histochem Cytochem
PubMed ID11101627
'To increase the sensitivity of fluorescence in situ hybridization (FISH) for detection of low-abundance mRNAs, we performed FISH on cryostat sections of rat hypothalamus with biotin-labeled riboprobes to leptin receptor (ObRb) and amplified the signal by combining tyramide signal amplification (TSA) and Enzyme-Labeled Fluorescent alkaline phosphatase substrate (ELF) methods. First, ... More
Immunofluorescence assay for the quantitative and qualitative evaluation of intracellular interleukin-8 in microtiter plates.
AuthorsWolff B, Zsak M, Rabeck C
JournalAnal Biochem
PubMed ID9025904
'In order to monitor the effects of drugs on interleukin-8 (IL-8) production by cells, a microtiter plate assay that determines four parameters simultaneously was established (i) levels of secreted IL-8 (supernatant ELISA), (ii) levels of intracellular IL-8 (cell ELISA), (iii) intracellular localization (fluorescence microscopy), and (iv) the amount of cellular ... More
AuthorsMendoza LG, McQuary P, Mongan A, Gangadharan R, Brignac S, Eggers M
JournalBiotechniques
PubMed ID10524321
'A new generation biochip is described as capable of supporting high-throughput (HT), multiplexed enzyme-linked immunosorbent assays (ELISAs). These biochips consist of an optically flat, glass plate containing 96 wells formed by an enclosing hydrophobic Teflon mask. The footprint dimensions of each well and the plate precisely match those of a ... More
Affinity capture of proteins from solution and their dissociation by contact printing.
AuthorsBernard A, Fitzli D, Sonderegger P, Delamarche E, Michel B, Bosshard HR, Biebuyck H
JournalNat Biotechnol
PubMed ID11533647
'Biological experiments at the solid/liquid interface, in general, require surfaces with a thin layer of purified molecules, which often represent precious material. Here, we have devised a method to extract proteins with high selectivity from crude biological sample solutions and place them on a surface in a functional, arbitrary pattern. ... More
Confocal multilaser focusing and single-laser characterization of ultraviolet excitable stains of cellular preparations.
AuthorsKahn E, Frouin F, Souchier C, Bernengo JC, Bruzzoni-Giovanelli H, Clément O, Frija G, Di Paola R, Calvo F, Linares-Cruz G
JournalCytometry
PubMed ID10754516
'BACKGROUND: The aims of this study were (1) to realign cellular preparations when spots and structures are excited by different lasers of a confocal laser scanning microscope (multilaser studies); (2) to avoid the use of realigment methods by selecting fluorochromes that can be excited by only one laser (single-laser experiments). ... More
Enzymatic quantification of cell-matrix and cell-cell adhesion.
AuthorsLöster K, Horstkorte R
JournalMicron
PubMed ID10568230
Adhesion assays are powerful tools to investigate the adhesive properties of cells. The quantification of cell adhesion enables determination of the capacity of cells to stick to a target, screening for novel adhesion involved binding molecules, exploration of structure-function relationships of adhesion molecules, evaluation of adhesion targets, and examination of ... More
Functional dissociation of the basolateral transcytotic compartment from the apical phago-lysosomal compartment in human osteoclasts.
AuthorsMeagher J, Zellweger R, Filgueira L,
JournalJ Histochem Cytochem
PubMed ID15872059
Tartrate-resistant acid phosphatase (TRAP) is essential for elimination of Staphylococcus aureus, the main infectious agent responsible for osteomyelitis. This in vitro study investigated uptake and processing of fluorescence-labeled S. aureus by human osteoclasts and dendritic cells. The cells were stained for TRAP and the acidic compartment using a fluorescence-based protocol. ... More
Osteoclast size is controlled by Fra-2 through LIF/LIF-receptor signalling and hypoxia.
AuthorsBozec A, Bakiri L, Hoebertz A, Eferl R, Schilling AF, Komnenovic V, Scheuch H, Priemel M, Stewart CL, Amling M, Wagner EF,
JournalNature
PubMed ID18548006
Osteoclasts are multinucleated haematopoietic cells that resorb bone. Increased osteoclast activity causes osteoporosis, a disorder resulting in a low bone mass and a high risk of fractures. Increased osteoclast size and numbers are also a hallmark of other disorders, such as Paget's disease and multiple myeloma. The protein c-Fos, a ... More
Photobleaching of arterial autofluorescence for immunofluorescence applications.
AuthorsKingsley K, Carroll K, Huff JL, Plopper GE
JournalBiotechniques
PubMed ID11314262
Immunohistochemical localization of low-level antigens in the arterial vasculature is complicated by the presence of complex molecules such as collagen, elastin, cholesterol, and fluorescent lipids that exhibit autofluorescence over a wide spectrum of wavelengths. UV irradiation of arterial vasculature has remained ineffective in preparing samples for immunofluorescent staining because of ... More
Laminin-5 induces osteogenic gene expression in human mesenchymal stem cells through an ERK-dependent pathway.
AuthorsKlees RF, Salasznyk RM, Kingsley K, Williams WA, Boskey A, Plopper GE
JournalMol Biol Cell
PubMed ID15574877
The laminin family of proteins is critical for managing a variety of cellular activities including migration, adhesion, and differentiation. In bone, the roles of laminins in controlling osteogenic differentiation of human mesenchymal stem cells (hMSC) are unknown. We report here that laminin-5 is found in bone and expressed by hMSC. ... More
Activation of NF-kappa B by XIAP, the X chromosome-linked inhibitor of apoptosis, in endothelial cells involves TAK1.
AuthorsHofer-Warbinek R, Schmid JA, Stehlik C, Binder BR, Lipp J, de Martin R
JournalJ Biol Chem
PubMed ID10807933
Exposure of endothelial and many other cell types to tumor necrosis factor alpha generates both apoptotic and anti-apoptotic signals. The anti-apoptotic pathway leads to activation of the transcription factor NF-kappaB that regulates the expression of genes such as A20 or members of the IAP gene family that protect cells from ... More
AuthorsMcBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS
JournalDev Cell
PubMed ID15068789
Commitment of stem cells to different lineages is regulated by many cues in the local tissue microenvironment. Here we demonstrate that cell shape regulates commitment of human mesenchymal stem cells (hMSCs) to adipocyte or osteoblast fate. hMSCs allowed to adhere, flatten, and spread underwent osteogenesis, while unspread, round cells became ... More
Elevated expression of endoglin, a component of the TGF-beta-receptor complex, correlates with proliferation of tumor endothelial cells.
AuthorsMiller DW, Graulich W, Karges B, Stahl S, Ernst M, Ramaswamy A, Sedlacek HH, Müller R, Adamkiewicz J
JournalInt J Cancer
PubMed ID10225446
Endoglin/CD105 is a membrane protein involved in the TGF-beta receptor signalling pathway. Endoglin expression has been reported to be selective for a few cell types, in particular endothelial cells, although a number of conflicting reports have been published. In this study, we performed a detailed analysis of endoglin expression in ... 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
In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide.
AuthorsBartlett RJ, Stockinger S, Denis MM, Bartlett WT, Inverardi L, Le TT, thi Man N, Morris GE, Bogan DJ, Metcalf-Bogan J, Kornegay JN
JournalNat Biotechnol
PubMed ID10835598
In the canine model of Duchenne muscular dystrophy in golden retrievers (GRMD), a point mutation within the splice acceptor site of intron 6 leads to deletion of exon 7 from the dystrophin mRNA, and the consequent frameshift causes early termination of translation. We have designed a DNA and RNA chimeric ... More
Fluorimetric determination of theophylline in serum by inhibition of bovine alkaline phosphatase in AOT based water/in oil microemulsion.
AuthorsJourquin G, Kauffmann JM
JournalJ Pharm Biomed Anal
PubMed ID9919959
Theophylline is an effective bronchodilatator used in the treatment of asthma which requires frequent control because of its narrow therapeutic index. Over the past decade much attention has been dedicated to the peculiar properties of the inner water pools of AOT (sodium 2-bishexyl-ethyl sulfosuccinate) microemulsions as enzyme microreactors, yet few ... More
Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays.
AuthorsCall DR, Brockman FJ, Chandler DP
JournalInt J Food Microbiol
PubMed ID11482571
Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of ... More
Semi-automated positional analysis using laser scanning microscopy of cells transfected in a regenerating newt limb.
AuthorsPecorino LT, Brockes JP, Entwistle A
JournalJ Histochem Cytochem
PubMed ID8666741
Limb regeneration in urodele amphibians such as the newt is a key system for investigating the positional identity of cells. The regenerate arises locally from blastemal cells, mesenchymal progenitors that normally give rise to structures distal to the amputation plane but which can be respecified (proximalized) by treatment with retinoic ... More
2-(2'-phosphoryloxyphenyl)-4(3H)-quinazolinone derivatives as fluorogenic precipitating substrates of phosphatases.
AuthorsHuang Z, Terpetschnig E, You W, Haugland RP
JournalAnal Biochem
PubMed ID1336935
Characterization of 2-(2'-phosphoryloxyphenyl)-4(3H)-quinazolinone (PPQ) derivatives as fluorogenic precipitating substrates of phosphatases is reported in this work. Soluble and colorless PPQ derivatives can be specifically hydrolyzed by acid and alkaline phosphatases into insoluble products, 2-(2'-hydroxyphenyl)-4(3H)-quinazolinone (HPQ) derivatives which appear as fluorescent precipitates in water. The fluorescence and precipitation of HPQ depend ... More
HGF inhibits BMP-induced osteoblastogenesis: possible implications for the bone disease of multiple myeloma.
AuthorsStandal T, Abildgaard N, Fagerli UM, Stordal B, Hjertner O, Borset M, Sundan A
JournalBlood
PubMed ID17138824
The bone disease in multiple myeloma is caused by an uncoupling of bone formation from bone resorption. A key difference between patients with and patients without osteolytic lesion is that the latter have fewer and less active osteoblasts. Hepatocyte growth factor (HGF) is often produced by myeloma cells and is ... More
A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity.
AuthorsCox WG, Singer VL
JournalJ Histochem Cytochem
PubMed ID10544217
We describe a high-resolution, fluorescence-based method for localizing endogenous alkaline phosphatase in tissues and cultured cells. This method utilizes ELF (Enzyme-Labeled Fluorescence)-97 phosphate, which yields an intensely fluorescent yellow-green precipitate at the site of enzymatic activity. We compared zebrafish intestine, ovary, and kidney cryosections stained for endogenous alkaline phosphatase using ... More
Simultaneous multianalyte ELISA performed on a microarray platform.
AuthorsWiese R, Belosludtsev Y, Powdrill T, Thompson P, Hogan M
JournalClin Chem
PubMed ID11468236
BACKGROUND: A logical progression of the widely used microtiter plate ELISA is toward a protein array format that allows simultaneous detection of multiple analytes at multiple array addresses within a single well. Here we describe the construction and use of such a multiplex ELISA to measure prostate-specific antigen (PSA), alpha1-antichymotrypsin-bound ... More
The ELF -97 phosphatase substrate provides a sensitive, photostable method for labelling cytological targets.
AuthorsParagas VB, Kramer JA, Fox C, Haugland RP, Singer VL
JournalJ Microsc
PubMed ID12000550
We compared fluorescent signals obtained with fluorescein conjugates and the ELF-97 (enzyme-labelled fluorescence) phosphatase substrate [2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone] in labelling cytological structures requiring high spatial resolution. Enzymatic cleavage of the ELF-97 phosphatase substrate yields an extremely fine precipitate that remains well localized to the site of enzymatic activity. This precipitate fluoresces bright ... More
Functional expression and characterization of the wild-type mammalian renal cortex sodium/phosphate cotransporter and an 215R mutant in Saccharomyces cerevisiae.
AuthorsBernhardt F, Schoner W, Schroeder B, Breves G, Scheiner-Bobis G
JournalBiochemistry
PubMed ID10521262
The wild-type and an R215E mutant of the rat renal cortex sodium/phosphate cotransporter type 2 (NaPi-2) were functionally expressed in the yeast Saccharomyces cerevisiae strain MB192, a cell line lacking the high-affinity endogenous H+/P(i) cotransporter. The expression of the mRNA molecules and corresponding proteins was confirmed by Northern and Western ... More
Fluid shear stress induces less calcium response in a single primary osteocyte than in a single osteoblast: implication of different focal adhesion formation.
AuthorsKamioka H, Sugawara Y, Murshid SA, Ishihara Y, Honjo T, Takano-Yamamoto T
JournalJ Bone Miner Res
PubMed ID16813522
The immediate calcium response to fluid shear stress was compared between osteocytes and osteoblasts on glass using real-time calcium imaging. The osteoblasts were responsive to fluid shear stress of up to 2.4 Pa, whereas the osteocytes were not. The difference in flow-induced calcium may be related to differences in focal ... More
Detection of endogenous and antibody-conjugated alkaline phosphatase with ELF-97 phosphate in multicolor flow cytometry applications.
AuthorsTelford W, Cox W, Singer V
JournalCytometry
PubMed ID11169576
BACKGROUND: The fluorogenic alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF(R)-97 phosphate, for Enzyme-Labeled Fluorescence) has been used primarily in microscope-based imaging applications to detect endogenous AP activity, antigens and various ligands in cells and tissues, and nucleic acid hybridization. In a previous study, we demonstrated the applicability of ELF-97 phosphate for ... More
Phosphate stress in cultures and field populations of the dinoflagellate prorocentrum minimum detected by a single-cell alkaline phosphatase assay
AuthorsDyhrman ST, Palenik B
JournalAppl Environ Microbiol
PubMed ID10388722
Alkaline phosphatase activity is a common marker of phosphate stress in many phytoplankton, but it has been difficult to attribute alkaline phosphatase activity to specific organisms or groups of phytoplankton in the field with traditional biochemical procedures. A new alkaline phosphatase substrate, ELF-97 (enzyme-labeled fluorescence), shows promise in this regard. ... More
Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate.
BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was ... More
The ELF-97 alkaline phosphatase substrate provides a bright, photostable, fluorescent signal amplification method for FISH.
We used the ELF-97 (Enzyme-Labeled Fluorescence) phosphatase substrate, 2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone, with alkaline phosphatase conjugates of streptavidin and appropriate antibodies to amplify signals from biotinylated and haptenylated hybridization probes. The dephosphorylated product, ELF-97 alcohol, is a bright yellow-green fluorescent precipitate optimally excited at approximately 360 nm, with emission centered at approximately 530 ... More
Detection of extracellular phosphatase activity at the single-cell level by enzyme-labeled fluorescence and flow cytometry: the importance of time kinetics in ELFA labeling.
Authors
JournalCytometry A
PubMed ID19051328
Variable inter and intraspecies alkaline phosphatase activity within single cells of revived dinoflagellates.
Authors
JournalISME J
PubMed ID33568788
RGS18 acts as a negative regulator of osteoclastogenesis by modulating the acid-sensing OGR1/NFAT signaling pathway.
Authors
JournalJ Bone Miner Res
PubMed ID17576169
Marked differences in local bone remodelling in response to different marrow stimulation techniques in a large animal.
Authors
JournalEur Cell Mater
PubMed ID34008855
Generation of the Compression-induced Dedifferentiated Adipocytes (CiDAs) Using Hypertonic Medium.