Article Category: IRDye Reagents

Use Near-Infrared Fluorescent Probes for Pharmacokinetics and Biodistribution Studies

In Vivo Imaging with NIR Fluorescent ProbesNon-invasive preclinical imaging methods are critical for development of imaging agents and targeted therapeutics. Pharmacokinetics is the study of what the body does to a drug with respect to biodistribution and clearance. Traditionally-used radiolabeled probes have limitations such as cost, access, and safety. Near-infrared (NIR) fluorescence imaging offers a powerful alternative to radiolabeled probes for pharmacokinetics and biodistribution studies. NIR fluorescent optical imaging agents can be used to image the whole animal over time. And, more than one agent can be tracked in the same animal if each agent is labeled with a spectrally-distinct fluorophore.

In this webinar, Dr Amy Geschwender examines several case studies from the literature, and discusses:

  • Why NIR fluorescent probes are widely used for in vivo imaging
  • How fluorescence imaging of excised tissues and tissue sections is used to examine biodistribution in more detail
  • How to measure serum half-life and % injected dose per gram with NIR fluorescent probes

This webinar features data from the Pearl® Small Animal Imaging System, which was recently honored by Frost & Sullivan, in addition to advancements in NIR technology. Click here to learn more about this award.

Visit our website to learn more about BrightSite™ Optical Imaging Agents and IRDye® infrared dyes that can be used for your pharmacokinetic and biodistribution studies.

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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Click Chemistry Reagents from LI-COR® for Biomolecule Labeling

Biomolecule labeling continues to be a cornerstone feature of many in vitro and in vivo biological experiments. Click Chemistry has recently emerged as a convenient, versatile, and reliable method for labeling a wide variety of molecules for applications ranging from biomarker isolation to assay development.
Click Chemistry Workflow
LI-COR now offers a portfolio of Click Chemistry reagents for copper-catalyzed and copper-free methods. These products offer researchers flexibility to choose the correct reagent for a diverse array of applications. LI-COR Click Chemistry reagents include IRDye® 800CW, IRDye 680RD, and IRDye 650 infrared fluorescent dyes labeled with DBCO, azide, or alkyne groups.

Click Chemistry utilizes pairs of reagents that exclusively react with each other and are effectively inert to naturally-occurring functional groups such as amines. Unlike affinity interactions such as streptavidin-biotin, Click Chemistry forges covalent bonds between the reacting partners to deliver stable bioconjugates.

Click Chemistry reactions can be categorized into two separate groups, copper-catalyzed or copper-free. Copper-catalyzed Click Chemistry is used for initiating reactions between azides and alkynes. These reactions are also known as Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC). Although they initiate and accelerate Click Reactions, copper catalysts are cytotoxic and inappropriate for use in living systems.

Watch this informative webinar on IRDye Infrared Dye Reagents for Click Chemistry.

Click Chemistry Reagents Labeled with DBCO Groups Allow for Copper-Free Biomolecule Labeling Reactions

LI-COR now offers Click Chemistry reagents for copper-catalyzed and copper-free methods. One group of products within this portfolio includes IRDye® infrared dyes labeled with DBCO groups, which can be used for copper-free methods.

The dibenzocyclooctyne group (DBCO) allows copper-free Click Chemistry to be done with live cells, whole organisms, and non-living samples. DBCO groups will preferentially and spontaneously label molecules containing azide groups (—N3). Within physiological temperature and pH ranges, the DBCO group does not react with amines or hydroxyls, which are naturally present in many biomolecules. Reaction of the DBCO group with the azide group is significantly faster than with the sulfhydryl group (—SH, thiol).
Click Chemistry Copper-Free Reaction

Click chemistry reagents with DBCO groups are available in 0.5 mg and 5 mg sizes for 3 dyes: IRDye 80CW, IRDye 680RD, and IRDye 650. IRDye 800CW DBCO also comes in a 50-mg pack size. For other sizes, contact LI-COR Custom Services.

Watch this 18-minute webinar to learn more about Click Chemistry applications and the new LI-COR® Click Chemistry reagents.

Create a Complete Molecular Imaging Workstation

pearltrilogybuildsystemCombining the Odyssey® CLx Infrared Imaging System with the Pearl® Small Animal Imaging System creates a versatile molecular imaging workstation for in vivo and in vitro imaging.

BrightSite™ Optical Imaging Agents or probes developed using IRDye® infrared dyes can be used for in vitro, in vivo, and tissue imaging. This technology offers researchers the ability to take research from the cell to the animal, all within one lab.

Odyssey CLx Infrared Imaging System Capabilities:

  • Cell-based assays (binding capacity, specificity, competition, etc.) for optical agent development
  • Histology and whole organ imaging for studying clearance and specificity
  • Simultaneous two-color detection for two targets or one target with sample normalization

Pearl Small Animal Imaging Capabilities:

Validation Workflow and Molecular Imaging WorkstationFigure 1. Validation and Use of an IRDye Fluorescent Probe. After probe labeling, in vitro cellular assays and microscopy are used to confirm specificity. The desired target is then imaged in animals. Excised organs and tissues> can be examined for more detailed localization of the probe. Animal image captured with Pearl Imaging System. A more comprehensive discussion of approaches for the development of fluorescent contrast agents has also been published. Reference: Kovar, et al. Anal Biochem 367(2007) 1-12.

Molecular imaging – achieved with near-infrared fluorescent technology from LI-COR!

NEW! IRDye® Goat Anti-Mouse IgM Secondary Antibodies from LI-COR®!

IRDye Dye-labeled Goat anti-Mouse AntibodiesOur IRDye secondary antibody line is growing! We have recently added IRDye Goat anti-Mouse IgM (μ chain specific) secondaries labeled with:

  • IRDye 800CW (PN 926-32280)
  • IRDye 680RD (PN 926-68180) or
  • IRDye 680LT (PN 926-68080).

Just like all of the LI-COR IRDye secondary antibodies, these are highly cross-adsorbed secondary antibody conjugates suitable for a variety of applications (see the table below).

IRDye 800CW secondary antibodies are the antibodies of choice for a wide variety of applications in the 800 nm channel (see the list below). IRDye 800CW secondary antibodies can be used for 2-color detection when multiplexed with IRDye 680RD or IRDye 680LT secondary antibodies.

IRDye 680RD secondary antibodies are the antibodies of choice for In-Cell Western Assay and Western blot applications in the 700 nm channel. These antibodies can be used for 2-color detection when multiplexed with IRDye 800CW secondary antibodies. These antibodies are our most universal use 700 nm channel antibodies. Start using IRDye 680RD first over other 700 nm dyes. Dilution working range 1:10,000 – 1:40,000.

IRDye 680LT secondary antibodies have been proven the brightest signal for Western blot detection in the 700 nm channel and are comparable to Alexa Fluor 680 secondary antibodies. Choose IRDye 680LT secondary antibodies to get high signal and for specific uses of detection in the 700nm channel. These antibodies are not recommended when getting up and running on system. Once established near-infrared protocols are optimized with IRDye 680RD, IRDye 680LT can be used to optimize signals in the 700 channel. Dilution range 1:20,000 – 1:40,000. Note: optimization may be required with IRDye 680LT.

Application IRDye 800CW
IRDye 680RD
IRDye 680LT
Western Blot
In-Cell Western™ Assay Not Recommended
On-Cell Western Assay Not Recommended
Protein Array
2D Gel Detection
Tissue Section Imaging
Small Animal Imaging Not Recommended
Virus Titration Assay Not Known Not Known
FRET-based Assay Not Known Not Known

Note: Now, as of December 15, 2014, you can also get 0.1 mg sizes of all of our IRDye dye-labeled secondary antibodies. Check out our complete listing here and our new filtering tool!

Use IRDye® Labeled Oligonucleotides for Safer, Faster Fluorescent Gel Shift Assays

The EMSA (electrophoretic mobility shift assay) is used to study protein:DNA complexes and interactions. Protein:DNA complexes migrate more slowly than unbound linear DNA on a non-denaturing gel, causing a “shift.”

Also called “gel shift” or “gel retardation” assays, EMSA can be used to analyze sequence-specific recognition of nucleic acids by proteins.

Traditional, radioactive EMSA protocols can be easily adapted to near-infrared fluorescence EMSA detection by using IRDye end-labeled oligonucleotides and imaging with the Odyssey® CLx or Odyssey Classic Infrared Imaging System, providing a safe and sensitive alternative.

Comparison of Detection Methods for Fluorescent Gel Shift Assay

For more information on the EMSA workflow and a sample protocol for infrared fluorescent mobility shift assays, visit our website.

In-Cell Western™ Assay Application: Response of COS-7 Cells to Hydroxyurea

Application: Detecting phospho-p53 in COS cells in response to Hydroxyurea

Example of In-Cell Western Assay: Effects of Hydroxyurea on phospho-p53 on COS-7 cells

In this In-Cell Western assay application, the response of COS-7 cells to increasing doses of hydroxyurea was measured by a specific antibody (Anti-phospho-p53 from Cell Signaling Technology, P/N 9286) that detects phosphorylated-p53 (Ser16). Total ERK1 was used for normalization. The image represents a 96-well two-color In-Cell Western with the 700 and 800 nm channels detecting phosphorylated-p53 (Ser16) and total ERK1, respectively. Background wells were incubated with secondary antibody but no primary antibody. IRDye® 680RD secondary antibodies were used for detection in the 700nm channel and IRDye 800CW secondary antibodies were usd for detection in the 800nm channel.

Dose response graph of % induction of p53 phosphorylation with hydroxyurea in COS-7 cells

The graph represents the average of four sets of quantitative data, demonstrating the percent induction of phosphorylated-p53 (Ser16). Plate-based assays such as this can be imaged on the Odyssey® CLx or Odyssey Sa Infrared Imaging System.

For more uses of In-Cell Westerns Assays, visit our website.

Now Use the Same Primary Antibody for Immunoprecipitation AND Western Blotting!

Quick Western Kit - IRDye 680RD, PN 926-68100

LI-COR® has just rolled out a new way that the recently-released Quick Western Kit – IRDye® 680RD (see Would you like to save at least 90 minutes the next time you do a Western blot?) can be used in your research.

Not only can the Quick Western Kit reduce Western blotting time by 90 min, the kit ALSO serves as a detection solution for post-immunoprecipitation samples by Western blot because it does not bind to denatured mouse monoclonal or rabbit monoclonal antibodies. The key benefit is the ability to use the same antibody for immunoprecipitation and post-immunoprecipitation detection by Western blot. Seriously, how cool is that!!??!!

Using the same primary antibody for IP and Western blotting with the Quick Western Kit

Figure 1. A431 cell lysates were immunoprecipitated overnight with a monoclonal antibody against p53. The resulting immunoprecipitates were separated by SDS-PAGE. Lane 1: Negative IP control; Lane 2: Test sample ; Lane 3: A431 cell lysate positive control. Western blotting was performed using the same p53 monoclonal antibody and incubated with IRDye 680RD Immunoprecipitation Detection Reagent.

Protocol: Detection of post-immunoprecipitation proteins by Western blot using the Quick Western Kit – IRDye 680RD

For more information, visit our website. Here’s the kit pack insert. To order this product (online ordering available in select countries), go to our ecommerce site.

Studying bone growth, changes, tumors? Try IRDye® BoneTag™ Optical Probes.

BrightSite Optical Imaging Agents
IRDye BoneTag optical imaging agents are tetracycline derivatives that incorporate into mineralizing bone. Structural imaging of bone can be used to more precisely localize an area of disease. A second disease-specific targeting agent with a spectrally-distinct fluorescent label can be used to localize and track disease (such as a tumor) in the same animal. When the two images are overlaid, bone structure is displayed in one color and the other target appears in a different color.

IRDye 680RD BoneTag and IRDye 800CW BoneTag are part of the ready-to-use BrightSite™ optical agents family and make it easy to begin animal studies immediately. These bright fluorescent agents are labeled with IRDye fluorophores for NIR fluorescence optical imaging, and they target a variety of disease characteristics. Simply administer the agent, then image with any small animal imaging equipment with appropriate 680 nm or 800 nm filter sets. No engineered cells or animals are needed.

IRDye BoneTag agent for imaging bone structure and remodeling

Figure 1. IRDye 680 BoneTag agent for imaging of bone structure and remodeling. Tetracycline-derived probe reveals skeletal structure, and signal is stable for weeks. Dorsal view of mouse imaged with IRDye 680 BoneTag. Image acquired with Pearl® Small Animal Imaging System.

We’ll be at AACR in Chicago, April 1 – 4, Booth 3800. Stop by and talk to us about how you can start your small animal in vivo imaging experiments today.