'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
Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging.
AuthorsDempsey GT, Vaughan JC, Chen KH, Bates M, Zhuang X,
JournalNat Methods
PubMed ID22056676
'One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes; the properties of the probes, including photons per switching event, on-off duty cycle, photostability and number of switching cycles, largely dictate ... More
Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection.
AuthorsBates M, Dempsey GT, Chen KH, Zhuang X,
JournalChemphyschem
PubMed ID22213647
'Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously ... 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
Phosphine quenching of cyanine dyes as a versatile tool for fluorescence microscopy.
AuthorsVaughan JC, Dempsey GT, Sun E, Zhuang X,
JournalJ Am Chem Soc
PubMed ID23311875
We report that the cyanine dye Cy5 and several of its structural relatives are reversibly quenched by the phosphine tris(2-carboxyethyl)phosphine (TCEP). Using Cy5 as a model, we show that the quenching reaction occurs by 1,4-addition of the phosphine to the polymethine bridge of Cy5 to form a covalent adduct. Illumination ... More
Pin1 promotes histone H1 dephosphorylation and stabilizes its binding to chromatin.
AuthorsRaghuram N, Strickfaden H, McDonald D, Williams K, Fang H, Mizzen C, Hayes JJ, Th'ng J, Hendzel MJ,
Journal
PubMed ID24100296
Histone H1 plays a crucial role in stabilizing higher order chromatin structure. Transcriptional activation, DNA replication, and chromosome condensation all require changes in chromatin structure and are correlated with the phosphorylation of histone H1. In this study, we describe a novel interaction between Pin1, a phosphorylation-specific prolyl isomerase, and phosphorylated ... 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
Doxorubicin-loaded protease-activated near-infrared fluorescent polymeric nanoparticles for imaging and therapy of cancer.
AuthorsYildiz T, Gu R, Zauscher S, Betancourt T
JournalInt J Nanomedicine
PubMed ID30464453
'Despite significant progress in the field of oncology, cancer remains one of the leading causes of death. Chemotherapy is one of the most common treatment options for cancer patients but is well known to result in off-target toxicity. Theranostic nanomedicines that integrate diagnostic and therapeutic functions within an all-in-one platform ... More
An Immune Atlas of Clear Cell Renal Cell Carcinoma.
AuthorsChevrier S, Levine JH, Zanotelli VRT, Silina K, Schulz D, Bacac M, Ries CH, Ailles L, Jewett MAS, Moch H, van den Broek M, Beisel C, Stadler MB, Gedye C, Reis B, Pe'er D, Bodenmiller B
JournalCell
PubMed ID28475899
'Immune cells in the tumor microenvironment modulate cancer progression and are attractive therapeutic targets. Macrophages and T cells are key components of the microenvironment, yet their phenotypes and relationships in this ecosystem and to clinical outcomes are ill defined. We used mass cytometry with extensive antibody panels to perform in-depth immune ... More
Super-resolution (SR) fluorescence microscopy, a class of optical microscopy techniques at a spatial resolution below the diffraction limit, has revolutionized the way we study biology, as recognized by the Nobel Prize in Chemistry in 2014. Stochastic optical reconstruction microscopy (STORM), a widely used SR technique, is based on the principle ... More
Cationic nanoparticle as an inhibitor of cell-free DNA-induced inflammation.
AuthorsLiang H, Peng B, Dong C, Liu L, Mao J, Wei S, Wang X, Xu H, Shen J, Mao HQ, Gao X, Leong KW, Chen Y
JournalNat Commun
PubMed ID30327464
Cell-free DNA (cfDNA) released from damaged or dead cells can activate DNA sensors that exacerbate the pathogenesis of rheumatoid arthritis (RA). Here we show that ~40?nm cationic nanoparticles (cNP) can scavenge cfDNA derived from RA patients and inhibit the activation of primary synovial fluid monocytes and fibroblast-like synoviocytes. Using clinical ... More
Small Molecule Optoacoustic Contrast Agents: An Unexplored Avenue for Enhancing In Vivo Imaging.
AuthorsLaramie MD, Smith MK, Marmarchi F, McNally LR, Henary M
JournalMolecules
PubMed ID30366395
Almost every variety of medical imaging technique relies heavily on exogenous contrast agents to generate high-resolution images of biological structures. Organic small molecule contrast agents, in particular, are well suited for biomedical imaging applications due to their favorable biocompatibility and amenability to structural modification. PET/SPECT, MRI, and fluorescence imaging all ... More
Improved Tumor Penetration and Single-Cell Targeting of Antibody-Drug Conjugates Increases Anticancer Efficacy and Host Survival.
AuthorsCilliers C, Menezes B, Nessler I, Linderman J, Thurber GM
JournalCancer Res
PubMed ID29217763
Current antibody-drug conjugates (ADC) have made advances in engineering the antibody, linker, conjugation site, small-molecule payload, and drug-to-antibody ratio (DAR). However, the relationship between heterogeneous intratumoral distribution and efficacy of ADCs is poorly understood. Here, we compared trastuzumab and ado-trastuzumab emtansine (T-DM1) to study the impact of ADC tumor distribution ... More
Monitoring protease activity in biological tissues using antibody prodrugs as sensing probes.
AuthorsVasiljeva O, Menendez E, Nguyen M, Craik CS, Michael Kavanaugh W
JournalSci Rep
PubMed ID32246002
Proteases have been implicated in the development of many pathological conditions, including cancer. Detection of protease activity in diseased tissues could therefore be useful for diagnosis, prognosis, and the development of novel therapeutic approaches. Due to tight post-translational regulation, determination of the expression level of proteases alone may not be ... More
Conversion of an Injectable MMP-Degradable Hydrogel into Core-Cross-Linked Micelles.
AuthorsNajafi M, Asadi H, van den Dikkenberg J, van Steenbergen MJ, Fens MHAM, Hennink WE, Vermonden T
JournalBiomacromolecules
PubMed ID31945299
In this study, a new type of injectable hydrogel called "HyMic" that can convert into core cross-linked (CCL) micelles upon exposure to matrix metalloproteinases (MMP's), was designed and developed for drug delivery applications. HyMic is composed of CCL micelles connected via an enzyme cleavable linker. To this end, two complementary ... More