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In Vivo Apoptosis Detection

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With the advent of cell permeant technology in combination with an increase in fluorescent technology, it has become possible to detect apoptosis in living animals. International Qualex Flow Cytometry Systems is introducing an in vivo caspase detection system (FLIVO*). FLIVO is an injectable fluorescent probe used to quantitate apoptosis in live animals. FLIVO is a direct stain; once labeled, tissues are ready for analysis through a window chamber, fluorescent microscope, or by flow cytometry following sacrifice and no further processing is necessary. FLIVO is very easy to use. Just inject it into the animal and let it circulate 30-45 minutes, FLIVO specifically binds to active caspases and remains trapped in the cells undergoing apoptosis. Any unbound reagent will be flushed back out of the cell with the natural circulation of the animal. 

 

International Qualex Flow Cytometry Systems has two FLIVO labeled probes  (red sulforhodamine, SR, or green carboxyfluorescein, FAM) available for use in whole animals.  To use the kit, reconstitute the reagent with 50uL DMSO, add 200uL filter-sterilized 1x injection buffer and inject 40uL into each mouse.  The small kits contain enough material to inject 6 mice.  Larger animals may require more reagent.  

  *The FLIVO product was developed by Immunochemistry Technologies, Bloomington, MN
In vivo caspase detection

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Figure 1a&b

Figure 1a&b. In vivo labeling of caspase-positive neurons reveals a remarkable difference in the extent of apoptotic cell death in the nucleus magnocellularis (NM) of control (Fig 1a) and deafferenced (Fig 1b) bird brains. Experimental procedure: remove the cochlea of 5-day-old chicks; survive for 6 hours; inject FAM-FLIVO into live animals 35 minutes prior to sacrifice; perfuse animals and fix the brain; vibrotome section the brain and mount. Cochlea was not removed in control animals. In the control bird (intact NM, left), a low level of background staining with FLIVO was detected uniformly in all cell bodies. In the experimental chick (right), nearly every auditory neuron in the deafferenced NM was brightly stained with FAM-FLIVO. FAM-FLIVO reveals that caspases are activated in every neuron of the NM just 6 hours after cochlear removal. Loss of stimulation from the cochlea, the auditory branch of the inner ear, induces a high level of apoptosis in the NM, second order auditory neurons in the chick brainstem. Data courtesy of Ms. Yuan Wang, University of Washington.

Effects of Chemotherapy
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Figures 2 a&b

Mammalian breast tumors were implanted into a murine breast tumor model to determine the effects of chemotherapy. Figure 2a, non-treated tumor. Figure 2b, treated animal. Following treatment, researchers were able to determine that the chemotherapeutic regime induced apoptosis reducing the implanted tumor.

In Vivo FLIVO kits available:

PRODUCT NUMBER
TEST SIZE
FLIVO6-FAM
6 mice
FLIVO6-SR
6 mice
FLIVO24-FAM
24 mice
FLIVO24-SR
24 mice

Manual

References

Griffin , R.J., Williams, B.W., Loren, M., Bischof, J.C., Olin, M., Lee, B.W., and Johnson, G. 2006. Novel use of highly specific poly-caspase inhibitor for in vivo detection of apoptosis in tumors treated with vascular targeting agents. Poster presented at the American Association for Cancer Research, April 2006.

Griffin , R., Williams, B.W., Olin, M., Lowen, M., and Song, C.W. 2005. Thermoradiotherapy enhancement with anti-vascular strategy. Poster presented at the Radiation Research Society, October 17, 2005. Morrow, T., Paulson, E. October 16, 2006. Diabetes-induced allodynia: correlation between behavioral intensity, duration of diabetes, and periaqueductal gray (PAG) activation. Poster presented at the Society for Neuroscience annual meeting, October 2006.

Olin, M., Roy , S., Peterson, P., and Moliter, T. 2006. In vivo model for the detection of morphine-induced apoptosis. Poster presented at the Society of Neuroimmunology Pharmacology (SNIP), April, 2006.

 

 

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