'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
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
Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling.
AuthorsKrutzik PO, Nolan GP
JournalNat Methods
PubMed ID16628206
'Flow cytometry allows high-content, multiparameter analysis of single cells, making it a promising tool for drug discovery and profiling of intracellular signaling. To add high-throughput capacity to flow cytometry, we developed a cell-based multiplexing technique called fluorescent cell barcoding (FCB). In FCB, each sample is labeled with a different signature, ... More
Noninvasive tracking of donor cell homing by near-infrared fluorescence imaging shortly after bone marrow transplantation.
AuthorsUshiki T, Kizaka-Kondoh S, Ashihara E, Tanaka S, Masuko M, Hirai H, Kimura S, Aizawa Y, Maekawa T, Hiraoka M,
JournalPLoS One
PubMed ID20559437
'BACKGROUND: Many diseases associated with bone marrow transplantation (BMT) are caused by transplanted hematopoietic cells, and the onset of these diseases occurs after homing of donor cells in the initial phase after BMT. Noninvasive observation of donor cell homing shortly after transplantation is potentially valuable for improving therapeutic outcomes of ... More
A Small Molecule Inhibitor of Polycomb Repressive Complex 1 Inhibits Ubiquitin Signaling at DNA Double-strand Breaks.
'Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in aberrant gene silencing in human cancers and is a potential target for cancer therapy. Here we show that 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. The drug also inhibits the accumulation ... More
Efficient cytosolic delivery of molecular beacon conjugates and flow cytometric analysis of target RNA.
AuthorsChen AK, Behlke MA, Tsourkas A,
JournalNucleic Acids Res
PubMed ID18503086
'Fluorescent microscopy experiments show that when 2''-O-methyl-modified molecular beacons (MBs) are introduced into NIH/3T3 cells, they elicit a nonspecific signal in the nucleus. This false-positive signal can be avoided by conjugating MBs to macromolecules (e.g. NeutrAvidin) that prevent nuclear sequestration, but the presence of a macromolecule makes efficient cytosolic delivery ... More
The antibody-mediated targeted delivery of interleukin-10 inhibits endometriosis in a syngeneic mouse model.
AuthorsSchwager K, Bootz F, Imesch P, Kaspar M, Trachsel E, Neri D,
JournalHum Reprod
PubMed ID21705369
Endometriosis is still a highly underdiagnosed disease, and the current medical and surgical treatment of endometriosis is associated with a high recurrence rate. This study investigates the use of derivatives of the human antibody F8, specific to the alternatively spliced extra-domain A of fibronectin (Fn), for the imaging and treatment ... More
AuthorsChow EK, Zhang XQ, Chen M, Lam R, Robinson E, Huang H, Schaffer D, Osawa E, Goga A, Ho D,
JournalSci Transl Med
PubMed ID21389265
Enhancing chemotherapeutic efficiency through improved drug delivery would facilitate treatment of chemoresistant cancers, such as recurrent mammary tumors and liver cancer. One way to improve drug delivery is through the use of nanodiamond (ND) therapies, which are both scalable and biocompatible. Here, we examined the efficacy of an ND-conjugated chemotherapeutic ... More
Dual functional molecular imaging probe targeting CD20 with PET and optical imaging.
AuthorsPaudyal P, Paudyal B, Iida Y, Oriuchi N, Hanaoka H, Tominaga H, Ishikita T, Yoshioka H, Higuchi T, Endo K,
JournalOncol Rep
PubMed ID19513512
The present study aimed to develop a monoclonal antibody (mAb)-based double functional probe for PET and near-infrared fluorescence (NIRF) targeting CD20 and to cross validate the targeting efficacy of this dual functional probe. NuB2, anti CD20 mAb was conjugated with Alexa Fluor 750 and 64Cu through DOTA chelator. PET and ... More
Synthesis and evaluation of near-infrared (NIR) dye-herceptin conjugates as photoacoustic computed tomography (PCT) probes for HER2 expression in breast cancer.
We are evaluating PCT imaging in conjunction with NIR dye labeled Herceptin antibody for noninvasive assessment of HER2 expression in tumors. Herceptin was labeled with Alexa Fluor-750 amine reactive dye for characterization of photoacoustic and fluorescence signals. Measurements were performed in solution and after incubation in cultured cell lines that ... More
Assessing the sensitivity of commercially available fluorophores to the intracellular environment.
AuthorsChen AK, Cheng Z, Behlke MA, Tsourkas A,
JournalAnal Chem
PubMed ID18700780
The use of fluorescence has become commonplace in the biological sciences, with many studies utilizing probes based on commercially available fluorophores to provide insight into cell function and behavior. As these imaging applications become more advanced, it becomes increasingly important to acquire accurate quantitative measurements of the fluorescence signal. Absolute ... More
Fluorescence lifetime imaging system for in vivo studies.
AuthorsHassan M, Riley J, Chernomordik V, Smith P, Pursley R, Lee SB, Capala J, Gandjbakhche AH
JournalMol Imaging
PubMed ID17711778
In this article, a fluorescence lifetime imaging system for small animals is presented. Data were collected by scanning a region of interest with a measurement head, a linear fiber array with fixed separations between a single source fiber and several detection fibers. The goal was to localize tumors and monitor ... More
In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment.
AuthorsSipkins DA, Wei X, Wu JW, Runnels JM, Côté D, Means TK, Luster AD, Scadden DT, Lin CP
JournalNature
PubMed ID15959517
The organization of cellular niches is known to have a key role in regulating normal stem cell differentiation and regeneration, but relatively little is known about the architecture of microenvironments that support malignant metastasis. Using dynamic in vivo confocal imaging, here we show that murine bone marrow contains unique anatomic ... More