IntegriSense on the ASK
- Products and catalog numbers
- Using IntegriSense in vivo
- Using IntegriSense in vitro
- Application notes and posters
Integrins are a family of transmembrane glycoproteins receptors that mediate the attachment between a cell and the tissues surrounding it. They are heterodimers containing two distinct chains known as the α and β subunits. A variety of different α and β subunits have been characterized in mammals and the integrins are named according to the identity of each of the subunits. For example, the integrin αvβ3 appears to play a critical role in tumor growth, metastasis and angiogenesis.
IntegriSense™ agents are fluorescent agents that specifically target the integrin αvβ3. They are comprised of a highly selective small molecule antagonist that binds integrin αvβ3 and a near-infrared (NIR) fluorophore. These agents have been developed to enable in vivo and in vitro visualization and quantification of integrin expression in tumor cells (as well as in neovasculature). They are highly effective in monitoring tumor growth, tumor angiogenesis, and treatment efficacy.
Figure 1: The integrin-targeted probe (IntegriSense 645) was synthesized by labeling a derivative of the αvβ3 antagonist with an amine-reactive fluorophore. It was designed in such a way to allow maximal tissue penetration and minimal absorption by physiological absorbers such as hemoglobin or water.
Products and catalog numbers
|Product||Catalog Number||Ex/Em wavelength (nm)||Molecular weight (g/mol)||Validated Experiments||Applications|
|IntegriSense 645||NEV10640||649/666||1250||In vivo/Ex vivo|
Flow cytometry & In vitro microscopy
|IntegriSense 680||NEV10645||675/693||1432||In vivo/Ex vivo|
Flow cytometry & In vitro microscopy
|IntegriSense 750||NEV10873||755/775||1278||In vivo/Ex vivo|
Flow cytometry & In vitro microscopy
Using IntegriSense in vivo
In Vivo Imaging
The recommended procedure for in vivo imaging with IntegriSense 645 is administration via intravenous injection and imaging 6-24 hours post injection. Imaging may be performed as early as 3 hours with some reduction in target signal/noise. IntegriSense 645 will clear from tissues after approximately 6-7 days. Repeat injection and imaging may be performed every 3 days for longitudinal studies. It is recommended that a pre-injection baseline image be taken prior to reinjection and imaging, unless injections are performed every 7 days.
The recommended procedure for in vivo imaging with IntegriSense 680 is administration via intravenous injection and imaging 3 – 48 hours post injection. Imaging at earlier time points (~3 hrs) is recommended when imaging the vasculature. Imaging at later time points (24-48 hrs) is recommended when imaging tumors to reduce background.
The recommended procedure for in vivo imaging with IntegriSense 750 is administration via intravenous injection and imaging 24 hours post injection. Imaging may be performed as early as 6 hours with some reduction in target signal/noise. IntegriSense 750 will clear from tissues after ~5 days. Repeat injection and imaging may be performed every five days for longitudinal studies. It is recommended that a pre-injection baseline image be taken prior to reinjection and imaging.
|IntegriSense||Route of Injection||Mouse Dose (25 g)||Rat Dose (250 g)||Blood t 1/2||Tissue t 1/2||Optimal imaging time||Optimal Re-injection Time (complete clearance)||Route of Metabolism/ background tissue||FMT and IVIS settings|
|IntegriSense 645||IV (IP)||2 nmol||6-20 nmol||10 min||48 h||6-24 h||6-7 d||Bladder, kidneys||FMT 635/655
IVIS 640/660 with spectral unmixing
|IntegriSense 680||IV (IP)||2 nmol||6-20 nmol||10 min||24 h||24 h||14 d||Kidneys, intestines||FMT 680/700
|IntegriSense 750||IV (IP)||2 nmol||6-20 nmol||30 min||24 h||24 h||4-6 d||Kidneys||FMT 750/770
Figure 2: A) Blood pharmacokinetic profile for IntegriSense 645 was assessed by injecting CD-1 mice (n = 3 per time point) intravenously and collecting blood at multiple times post injection. B) Tissue samples were collected at the peak imaging time point (24 hours) and assessed by planar imaging. Mean counts/energy for each tissue were determined as a measure of tissue brightness. Tissue samples from tumors show that highest intensity of signal.
Figure 3: 4T-1 tumor-bearing mice were injected intravenously with 2 nmol IntegriSense 645. A) A group of mice were pre-injected (5 min before) with the specific competitor compound 5f and imaged by FMT 24 hours by 2D planar (inserts) and 3D tomographic imaging using the FMT® 2500. The panels show three representative mice per group. B) The tomographic imaging datasets were used to quantify tumor region fluorescence associated with integrin expression. The tumor volumes were determined using caliper measurements. C) The mice were imaged between 3 and 144 hours after injection with IntegriSense 645. The insert pictures show a representative mouse imaged at the different time points. The dashed line indicates the background fluorescence measured in control tissues.
Frozen Tissue Protocol
We have validated IntegriSense agents for use with frozen tissue samples. Here is a brief protocol with a recommended concentration of agent to use:
- Freeze tumor/tissue (without agent) and section 5-10 µm by cryostat.
- Incubate with 1 µM IntegriSense agent at 37ºC for 10-30 min.
- Wash 2x with PBS.
- Mount with anti-fade reagent.
- Fluorescence microscopy filter: Cy5 (IntegriSense 680, IntegriSense 645), or Cy5.5 or Cy7* (IntegriSense 750)
Using IntegriSense in vitro
IntegriSense agents enable imaging of tumors and neovasculature in a range of oncology applications.
Flow cytometry and in vitro microscopy
We have validated HER2Sense 645 for use with fluorescence microscopes and flow cytometers. Here is a brief protocol with a recommended concentration of agent to use:
- Culture cells in standard TC plate or chamber slide.
- Incubate cells with 0.02-0.5 μM IntegriSense agent for 30 min at 37 °C.
- Wash 1-2x with PBS. For flow cytometry, detach and resuspend cells in PBS.
- Flow cytometry filter settings: 712/21 (IntegriSense 645, IntegriSense 680), 780/60 (IntegriSense 750)
- Fluorescence microscopy filter: Cy5 (IntegriSense 645), Cy5.5 (IntegriSense 680), Cy5.5 or Cy7* (IntegriSense 750)
In Vitro Applications:
Figure 4: Raw 264.7 mouse macrophages or 4T-1 mouse breast adenocarcinoma cells were incubated with 0.25 μM of IntegriSense 645. Compound 5f is a specific competitor and 25 μM of it was added 15 minutes before IntegriSense 645 in control samples. A) Cells were then analyzed by fluorescence microscopy and B) flow cytometry using appropriate lasers and filters for detection at 645 nm wavelength. As shown in this figure, both Raw 264.7 and 4T1 cells are labeled by the IntegriSense 645 (red), while cells containing the specific competitor have significantly less signal.
Application notes and posters
- Poster: In Vivo Quantification of Integrin-Targeted and Protease-Activated Imaging Agents in Response to Anti-Angiogenic Therapy Using Quantitative Fluorescence Tomography
Q. Do IntegriSense agents cross the blood brain barrier?
A. IntegriSense has only been shown to cross into the brain in models where the blood-brain barriers are jeopardized (or compromised).
Q. Have there been any side-effects observed in the animals injected with IntegriSense?
A. None. Hundreds of animals injected without any side-effects.
Q. Can you aliquot and freeze IntegriSense 680/750 to make it last longer than the recommended 10 days?
A. It is not recommended??
Please visit our Citations Library for references using IntegriSense 645 on the IVIS or on the FMT.