Immunochemistry Technologies, LLC

Use ICT's detection kits:
in vivo for live animal studies with FLIVO™
in vitro to study cultured cells with FLICA™

Assess chemotherapy in live animal models (FLIVO™)

Figure 1a: mouse window chamber model

Contol FSaII has low level of apoptosis, FLIVO cat.#981
Figure 1b: live tumor before treatment. Not much killing; there is a low level of green fluorescence.

Treated FSaII tumor has a high level of apoptosis, FLIVO cat.#981

Figure 1c:live tumor after treatment. Yes, lots of killing (high level of green fluorescence)

Researchers can now assess chemotherapy in live animals using ICT’s new FLIVO™ fluorescent in vivo apoptosis detection kits. FLIVO™ is an injectable fluorescent probe used to quantitate caspase activity in live animals. It is a direct stain: once labeled, tissues are ready for analysis and no further processing is necessary. FLIVO™ is very specific. Only active caspase enzymes will covalently bind with the reagent, therefore only cells undergoing apoptosis will fluoresce. This makes FLIVO™ an ideal reagent to assess the efficacy of chemotherapy. FLIVO™ is very easy to use. Just inject it into the animal and let it circulate 30-45 minutes. Dying apoptotic tumor cells fluoresce green (Figure 1) or red (Figure 3), and can be counter-stained with other reagents (such as DAPI). Read the pdf

Figure 1: 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 (left) 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 (right) 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.

Quanititate the level of apoptosis with FLIVO.
Figure 2

Figure 2: Cell suspensions were made from excised FSaII fibrosarcoma tumors (like those in Figure 3) 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.

SCK mammary tumor, control animal show little apoptosis.

SCK mammary tumor, test animal show a lot of apoptosis.

Figure 3a: control animal Figure 3b: test animal

Figure 3: These pictures were taken of live SCK mammary tumors growing inside 2 different mice after injection with red SR-FLIVO™. The control mouse (Figure 3a) received a placebo while the test mouse (Figure 3b) 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 green FAM-FLIVO™, excised the tumor, trypsinized the cells, and analyzed them on a flow cytometer (Figure 2).

Figure 4: 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 (Figure 4, 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 (Figre 4, 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).

Assess the response to treatment in cultured tumor cells (FLICA™)

ICT's FLICA™ kits make it easy to assess the response to treatment in cultured tumor cells. With FLICA™, you will know if the cells are alive, in early apoptosis, late apoptosis, or necrosis. Use ICT's general poly caspases FLICA™ kits in red and green to assess treatment to detect all caspases, or use ICT's other kits to detect specific caspase 1, 2, 3&7, 6, 8, 9, 10, or 13 (Figure 8).It's easy. Just add the reagent to the media, let it incubate 1- 4 hours, wash the cells and read with a fluorescence plate reader, microscope, or flow cytometer. Apoptotic cells fluoresce red or green; necrotic cells fluoresce red. Read the pdf

HL60 in early apoptosis is green. HL60 in late apoptosis is green and red. HL60 in necrosis is red.
HL-60 cells
Figure 5a Figure 5b Figure 5c
Early apoptosis Late apoptosis Necrosis

Flow cytometry graph of apoptosis vs. necrosis using FLICA.
Figure 5d: Apoptosis vs Necrosis

Figure 5. HL-60 cells (human promyelocytic leukemia) were treated with a drug, then stained with FAM-FLICA™ and Propidium Iodide (both included in kit #92) and analyzed on a scanning laser cytometer. Cells in early apoptosis fluoresce green with FAM-FLICA™ (Figure 5a). Cells in late apoptosis are dually stained with FAM-FLICA™and PI: they fluoresce green (they have active caspases) and red (the cell membrane has permeabilized, Figure 5b). Necrotic cells fluoresce red (Figure 5c). Unstained live cells do not fluoresce (Figure 5d, quadrant D). In this experiment, the drug triggered the caspase cascade rather than being overtly toxic to the cells. Data courtesy of Dr. Zbigniew Darzynkiewicz of the Brander Cancer Center.

4 out of 5 Jurkat cells are apoptotic (Poly Caspases Kit, # 92)

Figure 6: Apoptotic Jurkat cells fluoresce green.

Figure 6: 4 out of 5 cells are apoptotic: Jurkat cells (T lymphocytes) were labeled with ICT's Poly-Caspases FLICA™ kit (cat.# 92). 4 cells fluoresce green (left), while the grey image (right) reveals 5 cells in the field. The 4 green cells are apoptotic = 80% of cells in this experiment had active caspases. The level of fluorescence can be quantified on a fluorescence plate reader or flow cytometer. Data courtesy of Dr. Brian W. Lee, ICT.

THP-1 cells dually stained with FAM-FLICA and Hoechst (cat. # 92).
Figure 7: Apoptotic THP-1 cells fluoresce green.

Figure 7: THP-1 cells (human monocytic leukemia) were dually stained with ICT’s green poly-caspases probe, FAM-FLICA™, and a blue DNA stain, Hoechst (included in kit #91). One cell (middle) has a high level of caspase activity as indicated by green fluorescence, therefore it is apoptotic. The lack of green and the concentrated blue DNA in the lower right cell indicate it is alive (not apoptotic). The upper left cell is necrotic (no green, scattered blue). Data courtesy of Dr. Brian W. Lee, ICT.

Use specific FLICA kits to detect individual caspases.

Figure 8: Apoptosis in U937 cells.

Figure 8: U937 cells (human leukemic monocyte lymphoma) were plated at 1x10^6 cells/mL in 200uL and exposed to a protein. Cells were then exposed to a control, TNF-alpha, or cycloheximide for 24 hours. 6 uL of diluted Caspase-8-FLICA™ or Caspase-9-FLICA™ was added for 1 hour. Cells were washed 3 times by spinning at 300g for 5 min., aspirating the supernatant, and replacing it with 200uL wash buffer.Cells were run on a flow cytometer. Using FLICA, the levels of caspase 8 and caspase 9 activity were quantified. Data courtesy of Jennifer Mitchell, Scripps.

Quantitate 4 population of cells with FLICA-HTS.

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800-829-3194 This page was updated on 04/04/2008 . Please contact us at 952-888-8788.