ICT’s Near-Infrared DyLight® Tracers are used in conjunction with Near Infrared DyLight® Free Dye Control Assays. The Near Infrared DyLight® Free Dye Control Assay uses the Near Infrared (NIR) fluorescent dye molecule. When injected, both the Free Dye Control and the Tracer reagents will generate a fluorescent signal, but only the Tracer should bind to active caspases and remain inside an apoptotic cell. The fluorescent signal generated from the Free Dye Control reveals where the fluorescent reagent may have become trapped inside the cell, tissues, or body of the animal without specifically binding with an active caspase. In the context of the overall experiment, this base level of fluorescence is considered background noise compared with the signal generated in animals that were injected with the NIR-DyLight® Tracer.
Near Infrared DyLight® Tracer Assays is a powerful method for assessing caspase activity in vivo. Like our FLICA® probes, but optimized for whole live animal imaging, NIR-DyLight® Tracers (sold separately) are non-cytotoxic fluorescent inhibitors of caspases. NIR-DyLight® poly caspase tracers contain the preferred binding sequence for most caspases, Val-Ala-Asp (VAD). This preferred poly caspase tripeptide binding sequence is labeled at the amino terminus end with *Dylight® 690 or 747 NIR label and linked at the carboxyl end to a fluoromethyl ketone (FMK) reactive entity.
Apoptosis is an evolutionarily conserved process of programmed cell suicide. It is centered on a cascade of proteolytic enzymes called caspases that are triggered in response to pro-apoptotic signals. Like most other proteases, caspases are synthesized as pro-form precursors that undergo proteolytic maturation, either autocatalytically or in a cascade by enzymes with similar specificity. Active caspase enzymes consist of two large (~20 kD) and two small (~10 kD) subunits that non-covalently associate to form a two heterodimer, tetrameric active caspase. Once activated, caspases cleave protein substrates leading to the eventual disassembly of the cell. Caspases have been identified in organisms ranging from C. elegans to humans. Mammalian caspases play distinct roles in both apoptosis and inflammation.
NIR-DyLight® Tracer Assays provide a simple yet accurate method to detect caspase activity in vivo. To label cells containing elevated levels of active caspases, inject the DyLight® Tracer intravenously and let it circulate. Because the Tracer is cell-permeant, it readily diffuses in and out of all cells it encounters as it circulates throughout the body. If there are active caspase enzymes inside a cell, the Tracer will form an irreversible covalent bond with a reactive cysteine on the large subunit of the caspase heterodimer, thereby inhibiting further enzymatic activity. The bound NIR-DyLight® Tracer will remain inside the cell if the cell membrane is intact. Any unbound DyLight® Tracer is removed from the circulation of the animal in about an hour. The remaining NIR fluorescent signal in the tissue is a direct measure of caspase activity that occurred at the time the reagent was injected. Apoptotic cells will retain a higher concentration of DyLight® Tracer and fluoresce brighter than non-apoptotic cells. There is no interference from pro-caspases or inactive forms of the enzyme. If the treatment is causing cell death via apoptosis, apoptotic cells will have an elevated level of caspase activity relative to non-apoptotic or negative control cells and fluoresce near-infrared with DyLight® Tracer.
An initial experiment may be necessary to determine when and how much NIR-DyLight® Tracer to inject based on the size of the animal, tissue type, experimental conditions, rate of apoptosis, and method of analysis. Generally, the longer DyLight® Tracer circulates, the lower the non-specific background signal; however, some apoptotic cells may be lost over time. After 60 minutes, most of the unbound NIR-DyLight® Tracer or Free Dye Control will have cleared the bloodstream. The bound NIR-DyLight® Tracer will remain inside an apoptotic cell and generate a positive signal if the cell membrane is still intact. Once the animals have been injected with NIR-DyLight® Tracer, they are ready for analysis and no further staining is necessary. Because NIR-DyLight® Tracers are a direct stain, it eliminates any false positives that may arise from manipulation of the tissue. This gives a true representation of the induction of apoptosis in vivo as a result of the experimental condition. Live animals may be analyzed in a whole animal imager, and /or tissues may be prepared and further analyzed by histological methods. Tissues labeled with NIR-DyLight® Tracer can be counter-stained with other reagents such as DAPI and fixed or frozen for future analysis. The fluorescence intensity can be quantified by excising the tissue and analyzing cells with a flow cytometer. NIR-DyLight® 690 Free Dye optimally excites at 690 nm and has a peak emission at 709 nm. NIR-DyLight® 747 Free Dye optimally excites at 747 nm and has a peak emission at 776 nm.
- Prepare samples and controls.
- Dilute 10X Injection Buffer 1:10 with 45 mL diH20.
- Reconstitute NIR-FLIVO 747 Free Dye with 50 μL DMSO.
- Dilute NIR-FLIVO 747 Free Dye 1:10 with 450 μL 1X Injec¬tion Buffer.
- Inject 50 μL intravenously.
- Let NIR-FLIVO 747 Free Dye circulate for 4 hours.
- Image the animal. NIR-FLIVO 747 Free Dye excites at 747 nm and emits at 776 nm.
- If not viewing directly, excise tissue and process samples for further analysis.
- Optional: label excised samples with additional stains, such as Hoechst 33342, or an antibody.
- If desired, fix excised samples.
- Analyze excised samples with a fluorescence microscope or flow cytometer.
FLIVO® is a registered trademark of ICT. *Dylight® is a registered trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.