Real time imaging of apoptosis in vivo
The following protocol is suitable for the non-invasive monitoring of apoptosis. This protocol is a guideline only and we recommend following the instructions for use provided with each product as concentration, preparation and excitation/emission may between each product.
Principle of the CAS-MAP™ probe
The CAS-MAP™ probe is injected intravenously (IV) and allowed to circulate. The low molecular weight and characteristics of the probe allow it to penetrate the cell membrane and the blood brain barrier/blood retinal barrier. Once the probe is inside the cell, it forms a covalent bond with the active catalytic site of caspases, which tags the active caspase with a fluorescent label. The bound reagent remains inside the cell membrane of apoptotic cells, while unbound reagent is removed from non-apoptotic tissue by the circulatory system, kidneys and liver. Apoptotic cells have more active caspase than control cells and therefore fluoresce brighter when labeled with the caspase probe.
User specific optimizations:
Each biological system has different properties and mechanisms, therefore optimization for your own system must be performed. The following conditions must be optimized:
- Amount of probe given based on the weight of your animals, the location of the target tissue to be stained in relation to the injection route, the vasculature to the target tissue. When dosed IV, the probe will enter the target tissue for staining by the blood stream. Some tumor applications may be better suited for direct injection to the tumor. We are happy to guide you for your specific application, feel free to ask us. We suggest a 300 nmol/kg dose, but this may be reduced or increased based on your system.
- The timing of probe administration. Since the probe labels active caspases inside the cell, each researcher must have an idea of the optimal timing for administration based on the system (model and drug treatment). For example if the drug treatment initiates apoptosis 24 hours after administration, the probe would be administered 24 hours post treatment and imaged. The benefit of the probe and live animal imaging is that you can monitor over a time course to optimize this in your system.
- In the in vivo environment, apoptotic cells will eventually be phagocytized so you will want to image the animal within an hour of the probe administration. The apoptotic cells will continue to fluoresce as long as the cell is intact and not digested by phagocytes.
- Expose the animals to the experimental condition (i.e. drug treatment). Be sure to include positive and negative controls.
- Prepare the Imaging probe immediately prior to use.
- At the time point desired, administer 1x CAS-MAP solution into the tail vein (or desired IV injection site).
- Allow the probe to circulate (30-60 minutes).
- Image the animal with a small animal imager using appropriate excitation and emission settings.
- For time course studies, the animal can be given the probe and imaged at the desired time points. The probe needs to circulate for 30-60 minutes prior to each time point desired.
- At the end of the study, the tissue may also be removed and imaged or processed for additional analysis/staining.