A high-throughput, cell-based screening method for siRNA and small molecule inhibitors of mTORC1 signaling using the In Cell Western technique.
AuthorsHoffman GR, Moerke NJ, Hsia M, Shamu CE, Blenis J,
JournalAssay Drug Dev Technol
PubMed ID20085456
'The mTORC1 pathway is a central regulator of cell growth, and defective mTORC1 regulation plays a causative role in a variety of human diseases, including cancer, tumor syndromes such as the tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), and metabolic diseases such as diabetes and obesity. Given the importance of ... More
Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry.
'Intracellular flow cytometry permits quantitation of diverse molecular targets at the single-cell level. However, limitations in detection sensitivity inherently restrict the method, sometimes resulting in the inability to measure proteins of very low abundance or to differentiate cells expressing subtly different protein concentrations. To improve these measurements, an enzymatic amplification ... More
Mps3p is a novel component of the yeast spindle pole body that interacts with the yeast centrin homologue Cdc31p.
AuthorsJaspersen SL, Giddings TH, Winey M
JournalJ Cell Biol
PubMed ID12486115
'Accurate duplication of the Saccharomyces cerevisiae spindle pole body (SPB) is required for formation of a bipolar mitotic spindle. We identified mutants in SPB assembly by screening a temperature-sensitive collection of yeast for defects in SPB incorporation of a fluorescently marked integral SPB component, Spc42p. One SPB assembly mutant contained ... More
Labeling of immune cells for in vivo imaging using magnetofluorescent nanoparticles.
AuthorsPittet MJ, Swirski FK, Reynolds F, Josephson L, Weissleder R
JournalNat Protoc
PubMed ID17406214
'Observation of immune and stem cells in their native microenvironments requires the development of imaging agents to allow their in vivo tracking. We describe here the synthesis of magnetofluorescent nanoparticles for cell labeling in vitro and for multimodality imaging of administered cells in vivo. MION-47, a prototype monocrystalline iron oxide ... More
Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates.
AuthorsBerlier JE, Rothe A, Buller G, Bradford J, Gray DR, Filanoski BJ, Telford WG, Yue S, Liu J, Cheung CY, Chang W, Hirsch JD, Beechem JM, Haugland RP, Haugland RP
JournalJ Histochem Cytochem
PubMed ID14623938
'Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, ... More
Surface-functionalized nanoparticle library yields probes for apoptotic cells.
AuthorsSchellenberger EA, Reynolds F, Weissleder R, Josephson L
JournalChembiochem
PubMed ID14997519
'We have developed techniques for the efficient synthesis and screening of small libraries of surface-functionalized nanoparticles for the recognition of specific types of cells. To illustrate this concept we describe the development of a nanoparticle that preferentially recognizes apoptotic Jurkat cells in a manner similar to the apoptosis-recognizing protein annexin ... More
Sensitive quantitative protein concentration method using luminescent resonance energy transfer on a layer-by-layer europium(III) chelate particle sensor.
'A particle-based protein quantification method was developed. The method relies on adsorption of proteins on particles and time-resolved fluorescence resonance energy transfer (TR-FRET). Layer-by-layer (LbL) particles containing europium(III) chelate donor were prepared. A protein labeled with an acceptor was adsorbed onto the particles and near-infrared energy transfer signal was detected ... More
Multiplexed DNA detection using spectrally encoded porous SiO2 photonic crystal particles.
AuthorsMeade SO, Chen MY, Sailor MJ, Miskelly GM,
JournalAnal Chem
PubMed ID19271746
A particle-based multiplexed DNA assay based on encoded porous SiO(2) photonic crystal disks is demonstrated. A
Tumor-specific detection of an optically targeted antibody combined with a quencher-conjugated neutravidin
AuthorsOgawa M, Kosaka N, Choyke PL, Kobayashi H,
JournalBioconjug Chem
PubMed ID19072537
In vivo molecular cancer imaging with monoclonal antibodies has great potential not only for cancer detection, but also for cancer characterization. However, the prolonged retention of intravenously injected antibody in the blood causes low target tumor-to-background ratio (TBR). Avidin has been used as a ... More
In vivo target-specific activatable near-infrared optical labeling of humanized monoclonal antibodies.
AuthorsOgawa M, Regino CA, Choyke PL, Kobayashi H,
JournalMol Cancer Ther
PubMed ID19139133
Imaging with labeled monoclonal antibodies may be useful in detecting, staging, and monitoring tumors. Despite their high affinity and specificity, a critical limitation of antibody imaging is the high background signal due to prolonged clearance from the blood, which reduces the tumor-to-background ratio. To address this problem, we developed a ... More
A generalized kinetic model for amine modification of proteins with application to phage display.
AuthorsJin X, Newton JR, Montgomery-Smith S, Smith G,
JournalBiotechniques
PubMed ID19317659
Amine modification of filamentous virions (phage particles) is widely used in phage display technology to couple small groups such as biotin or fluorescent dyes to the major coat protein pVIII. We have developed a generalized kinetic model for protein amine modification and applied it to the modification of pVIII with ... More
In vivo optical molecular imaging of vascular endothelial growth factor for monitoring cancer treatment.
AuthorsChang SK, Rizvi I, Solban N, Hasan T,
JournalClin Cancer Res
PubMed ID18593993
PURPOSE: Vascular endothelial growth factor (VEGF) expression is a critical component in tumor growth and metastasis. Capabilities to monitor VEGF expression in vivo can potentially serve as a useful tool for diagnosis, prognosis, treatment planning, monitoring, and research. Here, we present the first report of in vivo hyperspectral molecular imaging ... More
Fluorescent DNA hybridization probe preparation using amine modification and reactive dye coupling.
AuthorsCox WG, Singer VL,
JournalBiotechniques
PubMed ID14740493
Fluorescent nucleic acid hybridization probes traditionally have been generated by enzymatic incorporation of dye-labeled nucleotides, even though incorporation efficiency is low and variable from dye to dye. Alternatively, 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate (aa-dUTP) is enzymatically incorporated to generate amine-modified DNA, which is then chemically labeled with an amine-reactive fluorescent dye. We optimized ... More
Dual probe with fluorescent and magnetic properties for imaging solid tumor xenografts.
AuthorsShan L, Wang S, Sridhar R, Bhujwalla ZM, Wang PC
JournalMol Imaging
PubMed ID17445503
A dual probe with fluorescent and magnetic reporter groups was constructed by linkage of the near-infrared (NIR) fluorescent transferrin conjugate (Tf(NIR)) on the surface of contrast agent-encapsulated cationic liposome (Lip-CA). This probe was used for magnetic resonance imaging (MRI) and optical imaging of MDA-MB-231-luc breast cancer cells grown as a ... More
In vitro and in vivo evaluation of Alexa Fluor 680-bombesin[7-14]NH2 peptide conjugate, a high-affinity fluorescent probe with high selectivity for the gastrin-releasing peptide receptor.
AuthorsMa L, Yu P, Veerendra B, Rold TL, Retzloff L, Prasanphanich A, Sieckman G, Hoffman TJ, Volkert WA, Smith CJ
JournalMol Imaging
PubMed ID17532883
Gastrin-releasing peptide (GRP) receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN), a 14-amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study ... More
Homogeneous dual-parameter assay for prostate-specific antigen based on fluorescence resonance energy transfer.
Three monoclonal antibodies (Mab) specific for prostate-specific antigen (PSA) were used to design a homogeneous dual-parameter immunoassay based on fluorescence resonance energy transfer (FRET). One antibody was labeled with terbium(III) chelate, which acted as a donor, and the other two antibodies were labeled with fluorescent acceptor dyes (either Alexa Fluor ... More
Normal human kidney HLA-DR-expressing renal microvascular endothelial cells: characterization, isolation, and regulation of MHC class II expression.
AuthorsMuczynski KA, Ekle DM, Coder DM, Anderson SK
JournalJ Am Soc Nephrol
PubMed ID12707403
Human, but not murine, renal peritubular and glomerular capillaries constitutively express class II major histocompatibility (MHC) proteins at high levels in normal human kidney. Expression of class II proteins on renal microvascular endothelial cells (RMEC) makes it available to circulating lymphocytes and imparts a surveillance capacity to RMEC for controlling ... More