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Are
you killing the tumor? |
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Find out with FLIVO™ to monitor the efficacy of chemotherapy.
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FLIVO™
is an injectable in
vivo detection reagent that labels apoptotic cells green
or red in just 30 minutes prior to sacrifice. If the 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. |
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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™
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no killing yet
apoptosis before treatment
a very low level of caspase activity
lots of killing
apoptosis after 3 hours of treatment
a very high level of caspase activity
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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.
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These pictures were taken
through a window chamber of 1 live breast cancer 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) was able to assess the efficacy of the drug using
FAM-FLIVO™: ATO induced apoptosis in most of the tumor cells.  |
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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 200uL filter-sterilized 1x injection buffer, and
inject 40uL 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!
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control
animal; 18% of tumor cells have active caspases
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test
animal; 39% of tumor cells have active caspases
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These pictures were taken of live 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) excised the tumor, trypsinized the cells, and analyzed them on a flow cytometer
(below).
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Cell suspensions were made from excised mammary tumors (like those
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.
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picture of kit
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Green
FLIVO™ kits contain: FAM-FLIVO™ reagent and injection
buffer. Cat.# 980, 25 tests. |
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picture of kit
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Red
FLIVO™
kits contain: SR-FLIVO™
reagent and injection buffer.
Cat.# 983, 100 tests. |
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FLIVO™
Poly (Pan) Caspase in vivo
Apoptosis Detection Kits
(green & red fluorescent probes) |
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Color
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small,
6 mice |
large,
24 mice |
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Price |
Cat
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Price |
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Green
FAM- FLIVO™ Kit
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980 |
$139.00 |
981 |
$389.00 |
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Red
SR- FLIVO™ Kit
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982 |
$159.00 |
983 |
$399.00 |
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FLIVO™
Kits:
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Use
whole animals
Label apoptotic tissues
before sacrifice so further manipulation of the tissues won't cause
artifacts.
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Easy
Just inject
into the animal; no lysis or permeabilization steps.
• Accurate
FLIVO™ will not stain necrotic nor healthy tissues.
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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.
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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)
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 200uL 1X injection buffer to the reagent.
5. Inject ~40uL 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 350uL injection buffer to the reagent,
then in step 5, inject 100uL into each animal.
Prices
& Catalog #s
Order
today 800-829-3194!
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| Check
out our other apoptosis kits:
FLICA™:
detect apoptosis in cultured cells in vitro by adding the reagent directly to the media.
Magic
Red™: detect apoptosis in real
time by watching color develop.
MitoPT™:
detect apoptosis via the mitochondrial membrane breakdown, correlating with
cytochrome-c release. Caspase
antibodies and enzymes:
use our purified reagents in the rest of your studies. |
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