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HypoxiSense on the ASK


Hypoxia is a pathological condition where a region of the body is deprived of oxygen. Hypoxia can be associated with a variety of disease states, such as angiogenesis, pulmonary disease and inflammation. In addition, hypoxia occurs in tumors because the disorganized vascular networks cannot deliver blood borne oxygen. During hypoxia, carbonic anhydrase IX (CA IX), a cell surface enzyme, is induced and therefore, CA IX expression is a good biomarker for these disease states. HypoxiSense 680 is a carbonic anhydrase IX (CAIX) targeted fluorescent in vivo imaging agent that can be used to image CAIX overexpression in response to hypoxia. For example, HypoxiSense 680 detects the tumor cell surface expression of carbonic anhydrase 9 (CA IX) protein, which increases in cervical, colorectal, and non-small cell lung tumors. Pairing HypoxiSense with optical fluorescent imaging technology allows non-invasive imaging and quantitation of hypoxia-related changes in the cell in vivo.

hypoxisensefig1.jpg
Figure 1: The CA IX targeted agent (HypoxiSense 680) was synthesized by coupling the NIR (near infrared) fluorochrome VivoTag-680 (VT680) to a derivative of the CA inhibitor acetazolamide.


Products and catalog numbers


ProductCatalog NumberEx/Em wavelength
(nm)
Molecular weight (g/mol)Validated ExperimentsApplicationsStorage and Stability
HypoxiSense 680NEV11070670/6851500In vivo/Ex vivo
Flow cytometry
In vitro microscopy
Oncology
Angiogenesis
Inflammation
Pulmonary
Technical Data Sheet


Using HypoxiSense 680 in vivo/ex vivo


The general recommended procedure for in vivo imaging with HypoxiSense 680 is administration via intravenous injection and imaging 24-48 hours post injection. HypoxiSense 680 may also be injected intraperitoneally and imaged 24 hours post injection. HypoxiSense 680 is ideally suited for detecting hypoxia-induced changes in CA IX expression.

View instructions on setting up an in vivo mouse experiment with HypoxiSense 680 and imaging on an IVIS or FMT system.

Route of InjectionMouse Dose (25 g)Rat Dose (250 g)Blood t 1/2Tissue t 1/2Optimal imaging timeOptimal Re-injection Time (complete clearance)Route of Metabolism/ background tissueFMT and IVIS settings
IV2 nmol6-20 nmol2 min12 h24 h6-7 dKidneysFMT 680/700
IVIS 675/720
hypoxisensefig2.jpg
Figure 2: HypoxiSense 680 clears from the bloodstream quickly, with a half-life of approximately 4 minutes. However, it accumulates within hypoxic regions in tumor tissue with a half-life of 6 hours.

hypoxisensefig3.jpg
Figure 3: In Vivo Imaging. To determine the optimal imaging time for HypoxiSense 680, HeLa tumor-bearing mice were injected intravenously with 4 nmol HypoxiSense 680 or a control agent and imaged at various times (1, 4, 6, 24, 48, and 72h) by FMT. A) Images showing tumor signals in mice injected with either HypoxiSense 680 or a control agent. B) FMT quantification of total pmols of fluorescence over time of HypoxiSense 680 or a control agent. Dotted line shows the peak time for the best signal to noise ratio. C) Tissue samples were collected at the peak imaging time point (24 hours) and were assessed by 2D epifluorescence imaging. The mean counts/energy for each tissue was determined as a measure of tissue brightness. Tumors and kidneys showed the highest accumulation of HypoxiSense 680.

hypoxisensefig4.jpg
Figure 4: In Vivo Imaging. HeLa tumor-bearing mice were injected intravenously with 2 nmol of HypoxiSense 680 and imaged 24 hours after injection. Control mice were intravenously injected with unlabeled acetazolamide (AZ, 10 mg/kg) CAIX inhibitor one hour prior to agent injection to test for selective binding of HypoxiSense 680 to CAIX. A) 2D epifluorescence images of representative mice receiving HypoxiSense 680 with or without AZ inhibitor. B) 3D FMT imaging and quantification of tumor fluorescence in the same mice from panel A.

hypoxisensefig5.jpg
Figure 5: In Vivo Imaging. The effect of a hypoxic environment on HypoxiSense 680 signal in tumors was assessed in HeLa tumor-bearing mice. Mice with HeLa tumors were exposed to either normal air (~21% O2) or placed in a sealed environmental chamber containing only 8% O2. After 24 hours, all mice were injected intravenously with 2 nmol of HypoxiSense 680 and then imaged 24 hours after intravenous injection (continuing housing under either normoxic or hypoxic conditions). A) A 2D epifluorescence picture comparing the mice under the two conditions. B) A 3D FMT image and quantification of the tumor fluorescence of HypoxiSense 680 under the two different conditions.

Ex Vivo Imaging:

hypoxisensefig6.jpg
Figure 6: Ex Vivo Imaging. To determine the tumor tissue localization of HypoxiSense 680 and a control agent, tumors were excised from HeLa tumor-bearing mice and fluorescence was assessed by microscopy. One hour prior to sacrifice, the mice were injected with pimonidazole (a gold standard method of labeling hypoxic tissue). Five minutes prior to sacrifice, the non-hypoxic tumor regions were visualized by injecting Hoechst perfusion agent. HypoxiSense 680 /Control (red); Hoechst (blue); Anti-Pimonidazole/Anti-CAIX Abs (green).


Using HypoxiSense 680 in vitro


HypoxiSense 680 enables imaging of CA-IX expressing tumor cells ex. HeLa and HT-29; HCT-116 and MDA-MB-231 (negative).

Flow cytometry and in vitro microscopy

We have validated HypoxiSense 680 for use with fluorescence microscopes and flow cytometers.  Here is a brief protocol with a recommended concentration of agent to use:

  1. Culture cells in standard TC plate or chamber slide.  To induce hypoxia, transfer cells to a modular incubator chamber infused with mixed low oxygen gas for 24 h.
  2. Incubate cells with 1 uM HypoxiSense 680 for 1 hr at 37 °C.
  3. Wash 2x with PBS. For flow cytometry, detach and resuspend cells in PBS.
  4. Flow cytometry filter settings: 712/21, Fluorescence microscopy filter: Cy5.5.

In Vitro Imaging:

hypoxisensefig7.jpg
Figure 7: Tumor cells were cultured under normoxic (20% O2) and hypoxic (1.0% O2)) conditions. The cells were then incubated with 1 μM HypoxiSense 680 for 1 hour at 37 °C. The red (left) indicates HypoxiSense 680 signal and the blue indicates DAPI nuclear staining. For blocking with Acetazolamide (AZ): Before adding the test agents, cells were pre-incubated with 100 μM of Acetazolamide for 1 hour. Agents binding to normoxic cells were all negative by microscopy (images not shown). Binding of HypoxiSense 680 and control agent to HeLa cells was then determined by fluorescence microscopy and flow cytometry (middle). An approximate Kd of HypoxiSense 680 binding to tumor cells was determined by incubation with different concentrations of HypoxiSense 680 and determining the concentration at which binding at half maximal (right).


FAQs


Q. Can I use HypoxiSense 680 in humans?

A. No, HypoxiSense 680 is intended for animal research only and not for use in humans.


Q.Is HypoxiSense 680 an antibody or based on a metabolic reaction?

A. Neither, it is a modified version of the CAIX inhibitor acetazolamide.


Q. Has HypoxiSense 680 been used for frozen tissue labeling?

A. This has not been tested.


Q. Can HypoxiSense 680 be used with cells expressing GFP?

A. There should be no cross-reactivity between the HypoxiSense 680 and GFP. The Excitation and Emission wavelengths for these two fluors are far enough apart.   


Citations


Please visit our Citations Library for references using HypoxiSense 680 on the IVIS or on the FMT.