FLIVO APOPTOSIS CASPASE DETECTION KITS FOR IN VIVO USE MONITOR CANCER TREATMENT

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large SR-FLIVO kit #983

use FLIVO in live animals
Are you killing the tumor?
Use FLIVO™ to monitor the efficacy of chemotherapy.

FLIVO™ is an injectable in vivo detection reagent that labels apoptotic cells green or red in just 30 minutes prior to sacrifice. If your treatment induces apoptosis in tumor cells, it is probably effective at killing the tumor. Let FLIVO™ guide you to the most effective treatment within hours of administration. 

 

Need to assess neurodegeneration?
Use FLIVO™ to label dying apoptotic neurons.
FLIVO™ is an injectable in vivo detection reagent that labels apoptotic neurons green or red in just 30 minutes prior to sacrifice. FLIVO™ crosses the blood-brain barrier and enters neurons. If the neuron is apoptotic and has active caspases, the neuron will fluoresce red or green (green FAM-FLIVO™ shown at left, counterstained with red Nissl after excision; see data and read the neuroscience page).
    Researchers can now quantify apoptosis in living animals using ICT’s new FLIVO™ fluorescent in vivo apoptosis detection kits. FLIVO™ is an injectable fluorescent probe used to quantitate apoptosis in live animals. It is a direct stain; once labeled, tissues are ready for analysis 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. Apoptotic cells fluoresce green or red. View cells and tissues directly through a window chamber system or other accessible cavity, or sacrifice the animal and analyze cells with a fluorescent microscope, plate reader, or flow cytometer (see splenocyte data and tissue data). The tissues can be fixed or frozen for future analysis; protect from light during storage and handling.
    Because FLIVO™ is cell-permeant, it diffuses in and out of cells as it circulates throughout the body.  If there is an active caspase enzyme
inside the cell, it will form an irreversible covalent bond with FLIVO™

Apoptosis before treatment 
no killing yet
apoptosis before treatment
a very low level of caspase activity

Apoptosis after treatment 
lots of killing
apoptosis after 3 hours of treatment
a very high level of caspase activity
and retain the green or red fluorescent signal within the cell.  Any unbound reagent will be flushed back out of the cell with the natural circulation of the animal.  FLIVO™ appears to clear the circulation of the animal in about an hour.  We have successfully detected apoptosis in tumor tissues, splenocytes, bone marrow, brain, and eye tissue in mice. The reagent crosses the blood-brain barrier.
     FLIVO™ is very specific. Only active enzymes will covalently bind with the reagent, therefore only cells undergoing apoptosis will fluoresce. Since the FLIVO™ reagents do not use antibodies, they will not react with pro-caspases or inactive forms of the enzymes. FLIVO™ is very sensitive and will even detect background apoptosis, typically occurring in 2-6% of cells. Unlike annexin, FLIVO™ will not accumulate in non-apoptotic tissues, nor will it non-specifically bind to thymus-derived cells. 
      ICT has 2 FLIVO™ kits to detect apoptosis in whole animals: chose red or green fluorescence.  ICT’s FLIVO™ reagents are comprised of 3 segments: a red (sulforhodamine, SR) or green (carboxyfluorescein, FAM) fluorescent label; a 3-amino acid peptide inhibitor sequence targeted by active caspases: valine, alanine, and aspartic acid (VAD); and a fluoromethylketone group (FMK) that allows the formation of a covalent bond with the active enzyme.  The green in vivo FAM-FLIVO™ probe excites at 492nm and emits at 520nm. The red in vivo SR-FLIVO™ probe excites at 565nm and emits at >600nm.

window chamber mouse
These pictures were taken through a window chamber of 1 live FSaII murine fibrosarcoma tumor growing inside a mouse. Before any treatment was administered, FAM-FLIVO™  was injected into the mouse to determine how many of the tumor cells were apoptotic: caspase-positive cells turn green, caspase-negative cells are dark. The first picture (top) was taken 45 minutes after FAM-FLIVO™ was injected. Very few of the cells turned green, indicating that most of the tumor is living. The mouse was then injected with a drug (arsenic trioxide, ATO) for 3 hours. FAM-FLIVO™ was again injected into the mouse, and the second picture (bottom) was taken 45 minutes later. Within a few hours of treatment, Dr. Robert Griffin (U of MN, now at the U of AR) was able to assess the efficacy of the drug using FAM-FLIVO™: ATO induced apoptosis in most of the tumor cells. Read the pdf

     Use FLIVO™ alongside labeled antibodies and other probes for dual-staining studies ex vivo.  The red SR-FLIVO™ reagent can be used with GFP-transfected cells. After labeling with FLIVO™, cells can be fixed, embedded, or frozen for storage. We recommend analyzing the tissues within 24 hours, as the fluorescent label will photobleach when exposed to light, however samples have been frozen for weeks and re-analyzed with equivalent results.
     To use the kit, reconstitute the reagent with 50uL DMSO, add 550uL filter-sterilized 1x injection buffer, and inject 100uL into each mouse.  FLIVO™ kits come in 2 sizes: small and large. The small kits contain 1 vial of FLIVO™, and large kits contain 4 vials (all FLIVO™ kits include injection buffer).  Based in our initial mouse model, 1 vial of reagent contains enough material to inject 6 mice.  Larger animals may require more reagent.  Set up an initial reagent titration experiment to determine of the amount of reagent that will work best for the size of your animal and tissue type. 
     FLIVO™ complements ICT’s FLICA™ in vitro apoptosis detection kits, which enable researchers to quantify apoptotic cells grown in culture. FLICA™ kits are available with green or red fluorescent probes to quantitate active caspase 1, 2, 3&7, 8, 9 10, 13, or all of them at once (poly caspases).

Don't set up another apoptosis experiment until you call us.  Order today 800-829-3194!

 
Red control mouse tumor Red test mouse tumor
control animal; 18% of tumor cells have active caspases

 

 test animal; 39% of tumor cells have active caspases

 
These pictures were taken of live SCK mammary tumors growing inside 2 different mice after injection with red SR-FLIVO™. The control mouse (left) received a placebo while the test mouse (right) was treated with ATO. 24 hours after treatment, both mice were injected intravenously with ICT’s red SR-FLIVO™ in the tail vein. Pictures were taken 30 minutes later. The control tumor exhibits some level of apoptosis as expected, whereas the ATO-treated mouse tumor exhibits a much higher level of apoptosis (it is brighter red). Areas with high levels of active caspases, and thus more bound fluorescence, appear as overexposed white spots in the image. Black areas are living tumor cells. To quantify the level of caspase activity and apoptosis, Dr. Robert Griffin (U of MN, U of AR) repeated the experiment with FAM-FLIVO™, excised the tumor, trypsinized the cells, and analyzed them on a flow cytometer (below). Read the pdf

 

FLIVO FC data
Cell suspensions were made from excised mammary tumors (like those pictured above) and analyzed on a flow cytometer. After labeling both tumors with FLIVO™, the level of apoptosis could be quantified: it doubled in SCK mammary tumors after treatment with ATO. 39% of tumor cells treated with ATO were apoptotic; 18% of untreated tumor cells were apoptotic.  Read the pdf

 
#980 small green FAM-FLIVO kit

 

 Small Green FLIVO™ kits contain: FAM-FLIVO™ reagent (1 vial) and injection buffer. Cat.# 980, $179, small (enough for 6 rats or mice).
#981 large green FAM-FLIVO kit

 

 Large Green FLIVO™ kits contain: FAM-FLIVO™ reagent (4 vials) and injection buffer. Cat.# 981, $489, large (enough for 25 rats or mice).
#982 small red SR-FLIVO kit

 

 Small Red FLIVO™ kits contain: SR-FLIVO™ reagent (1 vial) and injection buffer. Cat.# 982, $184, small (enough for 6 rats or mice).
#983 large red SR-FLIVO kit Large Red FLIVO™ kits contain: SR-FLIVO™ reagent (4 vials) and injection buffer. Cat.# 983, $499, large (enough for 25 rats or mice). 
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.

FLIVO™ Poly (Pan) Caspase in vivo
Apoptosis Detection Kits
(green & red fluorescent probes)

Color

small

large
Cat #   Price Cat #   Price
 

Green FAM- FLIVO™ Kit

 980 $179 981 $489
 

Red SR- FLIVO™ Kit

 982 $184 983 $499

 
Non-invasive image of apoptosis in mice.
FLIVO™ allows you to non-invasively image apoptosis in whole live animals. Human colon carcinoma COLO205 cells were injected S/C into female nude mice (left, tumor circled). After 27 days, animals were treated with a control or TRAIL at 25 mg/Kg (right), then injected with SR-FLIVO™. TRAIL induces apoptosis within 1 hour, and the level of apoptosis significantly increased by 4-hours (right) and 24-hours post-treatment (data not shown). COLO205 tumor cells are being killed - they fluoresce brightly with SR-FLIVO™. Data courtesy of Dr. Peter Lassota, Caliper Life Sciences / Xenogen (preliminary data without systems optimization shown).

Frequently Asked Questions
see below

FLIVO™ Kits:
• Use whole animals Label apoptotic tissues before sacrifice so further manipulation of the tissues won't cause artifacts. 
• Easy Just inject into the animal; no lysis or permeabilization steps.
• Accurate FLIVO™ will not stain necrotic nor healthy tissues. 
• Reliable Only cells with active caspase enzymes fluoresce; there is no interference from pro-caspases or inactive forms of the enzyme.  
• Fast Let the reagent circulate 30-45 minutes. 
• Direct Active caspases covalently bind to FLIVO™ which is fluorescent.  
 • Sensitive Detects background levels of apoptosis in controls. 
 • Safe Use red or green fluorescent probes instead of radioisotopes. 
 • Quantitative Analyze animals with a fluorescence microscope, whole animal imaging system, plate reader, or flow cytometer.
  

Data:
 • Experimental data summary sheet
• Apoptosis detected in thymus, bone marrow, other tissues
• Mammary tumors become apoptotic when treated with ATO
• Splenocytes become apoptotic after morphine treatment
• Window chamber model of apoptosis detection
• Data and protocol in the kit manual (call us 800-829-3194)
References

Sample protocol:
 1. Expose your animals to your experimental condition, and create positive and negative controls if possible. 
2. Reconstitute the reagent with 50uL DMSO to form the stock concentrate (which can be frozen for future use). 
3. Dilute the injection buffer 1:10 with diH2O and sterilize by filtration.
4. Add 550uL 1X injection buffer to the reagent. 
5. Inject ~100uL of diluted reagent into each mouse. 
6. Let circulate 30-45 minutes. 
7. Examine tissues under a fluorescent microscope, or sacrifice and excise cells.
8. If desired, label cells with an additional stain, like 7AAD or labeled antibody.
9. If desired, fix, embed, or freeze cells. 
10. Analyze cells with a fluorescent microscope, plate reader or flow cytometer.

This protocol may be adjusted depending on your animal and target tissues.  For example, to increase the dose of FLIVO™ given to each animal, change steps 4 and 5.  In step 4, add 550uL injection buffer to the reagent (creating 600 uL of injection solution), then in step 5, inject 100uL into each animal.


Prices & Catalog #s

 

 

 

Order today!  Just call 1-800-829-3194

or download our simple order sheet (Word) or PDF
main: 952-888-8788    
fax: 952-888-8988

 

 

Frequently Asked Questions 
1. Can I inject more than 40 uL into the animal? Yes. Simply dilute the stock concentrate with more injection buffer and divide the final volume by 6 (as each vial of reagent contains enough for 6 small animals). For example, reconstitute FLIVO™ with 50 uL DMSO (to form the stock concentrate), and dilute this with 550 uL injection buffer. This yields 600 uL of injection solution - inject 100 uL per mouse. 
2. How many animals can I test with 1 vial? Each vial of reagent is designed for 6 mice weighing about 30 grams. Larger animals may require more reagent. We have not calculated an exact quantity of FLIVO™ needed per gram per animal.
3. Can I inject FLIVO™ intraperitoneal (IP) instead of intravenously (IV)? We do not recommend IP injections: the reagent simply does not get access to the cells. When injected IP, or at the site of the tissue, the reagent will penetrate and label apoptotic cells nearby through several layers, but it may not permeant all the target tissues. Site-injections may be satisfactory for some experiments. We find that the reagent works best when pumped through the animal in the blood stream. 
4. Why does it need to circulate for 30-60 minutes? During the circulation period, the reagent enters and exits each cell with the pumping action of the blood system. FLIVO™ is cell-permeant crosses the cell membrane automatically. If a cell has active caspases, the caspase enzyme will form a covalent bond with the reagent. This complex is now simply too big to cross the cell membrane and leave the cell and the fluorescence is captured within the cell. If the cell does not have active caspases, the unbound FLIVO™ will be flushed out of the cell and continue to circulate throughout the body. FLIVO™ will be removed from the circulation in about an hour. After this time, any caspase-positive cells may begin to break down leading to a lower fluorescent signal. You will see an optimal amount of fluorescence in about 30-45 minutes. Continued circulation of 60 minutes may reduce any non-specific background signals. Circulation periods of longer than 60 minutes may be unnecessary. 
5. How long does it take to clear the body and where does it go? FLIVO™ will clear the circulation in about 1 hour. The reagent appears to be filtered out through the liver and kidneys, and possibly end up in the intestines, but more research is necessary to validate these theories. 
6. Can I use FLIVO™ with other animals besides mice? Yes. FLIVO™ can be used with other animals. We have used it with mice, rats, chicks, and sparrows. 
7. How do I read the tissues? With green or red fluorescence. You can examine the tissues directly through a window-chamber models system, or excise the tissues and read with a fluorescence microscope, or quantify cell death with a flow cytometer. We have done some preliminary experiments using a Xenogen IVIS instrument (a whole-body imager) which suggests that the reagent may be detected through the intact animal. Please contact ICT for the latest update. 
8. How long will the fluorescence signal last? As long as the cells are protected from light, the signal will remain. Ideally, samples should be read within 24 hours. We have frozen brain samples that are still bright after 3 months of storage. 
9. Can I perfuse the animal? Yes. Sacrifice the animal and prepare tissues as usual: the animal can be perfused; the tissues can be fixed or frozen and thin sections made; or the cells can be put in suspension. Protect from light while handling. 
10. Can I do any other secondary labeling? Yes. FLIVO™ is available in 2 colors: red or green fluorescence so you can do dual-staining studies. FLIVO™ works very well with other blue, red, and green labels such as DAPI, GFP, Hoechst, PI and 7-AAD. You can also do a Western with FLIVO™-labeled cells. Protect from light while handling. 
11. Is FLIVO™ specific for neurons or cancer? No. The reagent enters and exits all types of cells. It is specific for caspase enzymes, no matter what type of cell they are in. You can label your specific cell with another marker, such as Nissl for neurons, or a labeled antibody for cancer cells. 
12. Does FLIVO™ cross the blood-brain barrier? Yes. We have looked at rats and sparrows and it enters the neurons without any special treatment. 
13. Does FLIVO™ cross the retinal-blood barrier? Yes. We have looked at mice and it labels retinal glial cells nicely without any special treatment. 
14. How does FLIVO™ get into the cell? FLIVO™ is cell-permeant. ICT has optimized the reagent to enter the cell without any lysis or permeabilization steps. 
15. Will I see any background fluorescence? You might. We have seen a low level of fluorescence throughout the animal, but can still easily and clearly distinguish caspase-positive cells from negative cells. In one case, we had high backgrounds throughout a frozen 20 micron section. The background was easily reduced by exposing the tissue to bright light under the fluorescence microscope for 15 minutes prior to reading the sample. We could still clearly distinguish caspase-positive from negative cells. We generally recommend protecting the cells from light, as the positive signal will eventually bleach out under strong fluorescence. Letting the reagent circulate longer than 60 minutes may help to reduce any non-specific background signals. 

Are you killing the tumor? postcard

Check out our other apoptosis kits:

in vitro analysis of whole living cells in culture, use FLICA™.

real-time analysis of caspase activity in culture, use Magic Red™.

detect mitochondrial membrane break-down which correlates with cytochrome-c release, use MitoPT™: 

Caspase antibodies and enzymes: use our purified reagents in the rest of your studies.

Order today!  Just call 1-800-829-3194

or download our simple order sheet (Word) or PDF
main: 952-888-8788    
fax: 952-888-8988
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Copyright 2008                                    This page was updated on 04/24/2008 . Please contact us at 952-888-8788.