Journal Articles Citing Use of Odyssey® or Pearl® Imaging Systems and Near-Infrared Fluorescence

The following are 4 journal references citing the use of either Odyssey or Pearl Imaging Systems.

Affibody-DyLight Conjugates for in vivoAssessment of HER2 Expression by Near-Infrared Optical Imaging.

Zielinski R, M Hassan, I Lyakhov, D Needle, V Chernomordik, A Garcia-Glaessner, Y Ardeshirpour, J Capala and A Gandjbakhche
Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
PLoS ONE 7(7): e41016 (2012). doi:10.1371/journal.pone.0041016

The HER2/neu gene is overexpressed in ~20% of invasive breast carcinomas. in vivo assessment of HER2 levels would aid development of HER2-targeted therapies and perhaps assist in selection of appropriate treatment strategies. This study describes HER2-specific probes for in vivo monitoring of receptor levels by near-infrared (NIR) optical imaging. Affibody molecules were labeled with DyLight750 dye, and affinity and specificity were confirmed in vitro. in vivo, Affibody-DyLight probes accumulated in HER2-positive breast cancer xenografts, but not in HER2-negative xenografts.

Fluorescent images were acquired at different time intervals after probe injection.
Fluorescent images were acquired at different time intervals after probe injection. Mouse bearing BT-474 xenograft tumor was injected with 10 µg HER2-Affibody-DyLight750 conjugate. Images were acquired every second for 1 minute with Pearl Impulse Imager (LI-COR Biosciences). doi:10.1371/journal.pone.0041016.s004

Animals were imaged with a custom NIR fluorescence-lifetime imaging system. The Pearl® Impulse Imager (LI-COR Biosciences) was used to monitor real-time accumulation of the Affibody probe in HER2-positive tumors during very early time points. Probe was injected during image acquisition, and images were captured every second for 1 minute. Probe accumulation in the kidney first, followed by tumor accumulation. Tumor fluorescence could still be detected 5 days after probe injection. This Affibody conjugate is useful for preclinical monitoring of HER2 status, and may have clinical utility.


Disruption of Kv1.3 Channel Forward Vesicular Trafficking by Hypoxia in Human T Lymphocytes

AA Chimote, Z Kuras, and L Conforti
Departments of Internal Medicine and Molecular & Cellular Physiology, University of Cincinnati, Cincinnati, Ohio
Journal of Biological Chemistry 287(3): 2055-67 (2012) DOI 10.1074/jbc.M111.274209

In solid tumors, hypoxia decreases immune surveillance. Kv1.3 channels on T lymphocytes are down-regulated by an unknown mechanism, inhibiting T cell function. The authors hypothesize that changes in membrane trafficking cause reduced expression of Kv1.3 at the cell surface. On-Cell Western cell based assays (Odyssey® Imager, LI-COR Biosciences) were extensively used to measure cell surface expression of Kv1.3.

Chronic hypoxia decreased cell surface expression of Kv1.3 in Jurkat cells. Inhibition of protein synthesis, degradation, or endocytosis did not block this effect. However, inhibition of forward trafficking in the trans-Golgi with brefeldin A (BFA) prevented hypoxia-induced reduction of Kv1.3 cell surface expression. Confocal microscopy confirmed retention of Kv1.3 in the trans-Golgi. Quantitative fluorescent Westerns (Odyssey Imager) demonstrated that expression of AP-1, which is required for clathrin-coated vesicle formation, is downregulated by hypoxia. These data indicate that chronic hypoxia disrupts clathrin-mediated forward trafficking of Kv1.3, thereby reducing immune surveillance by T cells.


Sequential Application of Anticancer Drugs Enhances Cell Death by Rewiring Apoptotic Signaling Networks

M Lee, A Ye, A Gardino, A Hheijink, P Sorger, G MacBeath, and M Yaffe
Dept of Biology, David H. Koch Institute for Integrative Cancer Research, Cambridge, Massachusetts, USA.
Cell 149:780-794 (2012). doi: 10.1016/j.cell.2012.03.031

Historically, standard treatments for human malignancies have been single drug therapies that cause DNA damage. Systems-based approaches and network analysis are now being used to examine how signaling can be re-wired by drug treatments that target dynamic network states. This study suggests that the timing and order of administration of certain drug combinations increases treatment effectiveness. Lee et al. pre-treated cells with epidermal growth factor receptor (EGFR) inhibitors, prior to DNA-damaging chemotherapy drugs.

Pre-treatment with erlotinib (an EGFR inhibitor) sensitized triple-negative breast cancers (TNBCs) to the DNA damage agent doxorubicin, and cell death increased by nearly 500%. Sensitization occurred only if the drugs were given sequentially. Transcriptional, proteomic, and computational analysis of signaling networks showed that dynamic network re-wiring was responsible for sensitization. Quantitative Westerns (Odyssey Imager; high-density, 48-sample blots) were used to monitor systems-level signaling dynamics. Erlotinib treatment made cells more susceptible to DNA damage by reactivating an apoptotic pathway that had been suppressed.


Investigation of Ovarian Cancer Associated Sialylation Changes in N-linked Glycopeptides by Quantitative Proteomics

V Shetty, J Hafner, P Shah, Z Nickens, and R Philip
Immunotope, Inc., Doylestown, Pennsylvania, USA
Clinical Proteomics 9:10 (2012) doi:10.1186/1559-0275-9-10.

CA125 is currently used as a biomarker for ovarian cancer, but is ineffective for detection of early stage disease. Previous research indicates that the level of sialic acid in total serum of ovarian cancer patients is elevated. Based on that idea, the authors suggest using N-linked sialyated glycopeptides as potential targets for early stage ovarian cancer biomarker discovery.

Shetty et al. used Lectin-directed Tandem Lableing (LTL) and iTRAQ quantitative proteomics to investigate N-linked sialyated glycopeptides, and identified 10 that were up-regulated in serum from ovarian cancer patients. Quantitative Western blot analysis of lectin-enriched glycoproteins (Odyssey Imager) was used to confirm the proteomic analysis. In ovarian cancer, increased sialylation of haptoglobin, PON1, and Zinc-alpha-2-glycoprotein was observed. Cancer-specific sialylation of glycopeptides may be a target for biomarker discovery.


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Quantitative Cell Signaling Analysis – What’s all the Buzz About?

In-Cell Western Assays for Quantitative Cell Signaling Analysis

What’s all this BUZZZZ you are hearing about being able to quantitate cell signaling in plate-based assays? If you are at AACR in Chicago this week, stop by Booth 3800 (LI-COR® Biosciences) and we can tell you all about the In-Cell Western™ Assay – and how you can use this method to quantitate signaling, look at levels of protein phosphorylation, perform RNAi studies, monitor gene expression levels, conduct cell proliferation assays, and more. Imaging can be performed on the Odyssey® CLx, Odyssey Classic, or the Odyssey Sa Infrared Imager (the Sa also has the option for automation and barcode reading). And, if you can’t make it to AACR, stay tuned here and I will be blogging about this topic over the next week or so.

Okay, let’s start at the beginning. So what – exactly – is an In-Cell Western Assay? Well, some call it a cytoblot. To others, it’s a cell-based ELISA or an In-Cell ELISA (ICE Assay). To LI-COR, it’s a In-Cell Western Assay (we call it an ICW, for short) and is a quantitative immunofluorescence assay performed in microplates (96- or 384-well format). It combines the specificity of Western blotting with the reproducibility and throughput of ELISA.

In a nutshell, the basic steps are:

  • Culture cells in microplates
  • Treat cells
  • Fix and permeabilize
  • Stain with primary antibodies – 1 or 2 protein targets per well
  • Stain with IRDye secondary antibody conjugates
  • Image microplate and quantify fluorescent signals from cell populations in each well
  • Quantify relative protein levels
  • Normalize to correct for well-to-well variation

That doesn’t sound too difficult, right? Of course, just like any scientific technique, there are things to keep in mind to make sure your experiment gives the best, clearest, most accurate and reproducible results it can. In the next posts, I’ll share some of the technical tips to keep in mind – plus examples of how your research colleagues have used In-Cell ELISAs in their published papers.

In the meantime, here is a brochure on near-infrared applications for the Odyssey Imaging Systems, which includes a little more info on the technique and some examples with data. We also have a video introduction to In-Cell Western Assays – for those that like the movies!

Updated October 6, 2016.