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Immunology > Apoptosis
You have problems with the TUNEL method? AbCys gives you the solution!
DETECTION OF APOPTOTIC CELLS : anti-ssDNA Antibody
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I. PRINCIPLE OF THE PROCEDURE :
Apoptosis assay described herein is based on the increased sensitivity of DNA in apoptotic cells to thermal denaturation. In this method DNA is denatured by heating in the presence of formamide and stained with monoclonal antibody (MAb) F7-26 specific to single-stranded DNA (ssDNA).
II.Advantages OF THE PROCEDURE :
- 1. Necrotic cells with a high level of double-stranded DNA breaks, demonstrated by TUNEL, do not react with the anti-ssDNA MAb.
- 2. Apoptotic cells with very low and high levels of DNA fragmentation are stained by formamide-MAb technique with a similar intensity.
- 3. MAb F7-26 is not species-specific and could be used to detect apoptotic cells in various species (mouse, rat and human cells were tested).
III.APPLICATIONS
A - FLUORESCENCE MICROSCOPY
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| A DNA Fluorochrome DAPI |
B Mab to ssDNA |
Fluorescence microphotographs (A and B) of MB-MDA-468 cells treated with staurosporine (inhibitor of protein kinase), heated in formamide, stained with Mab F7-26 and counterstained with DNA fluorochrome DAPI. The same field is shown after UV excitation for DAPI (left panel) and visible light excitation for fluorescein-labeled antibody (right panel).
Note that only 3 apoptotic cells with chromatin condensed at the nuclear periphery are stained with the Mab. Magnification, X1000
B - FLOW CYTOMETRY.
a. MAB TO ssDNA.
Fluorescence distributions of MB-MDA-468 cells heated in formamide and stained with MAb F7-26 generated on a flow cytometer. Apoptosis was induced by staurosporine and necrosis was induced by sodium azide or hyperthermia. Note that apoptotic cells are intensely stained with the MAb, while fluorescence profiles of necrotic and control cells are similar.
b. TUNEL.
Fluorescence distributions of MB-MDA-468 cells stained with TUNEL generated on a flow cytometer. Apoptosis was induced by staurosporine and necrosis was induced by sodium azide or hyperthermia. Note that apoptotic and necrotic cells are stained.
C - DETECTION OF APOPTOSIS IN TISSUE SECTIONS.
Small intestine of mice treated with DNA synthesis inhibitor, hydroxyurea and thymus of mice treated with hydrocortisone were fixed in formalin. Thin sections of tissues prepared with standard histological techniques were heated in formamide and stained with MAb F7-26.
Apoptotic cells with condensed chromatin and fragmented nuclei in the crypts of the small intestine from hydroxyurea-treated mice were stained with the Mab (Fig C). There was no staining of cells in the crypts of untreated animals (Fig D) Since crypts contain large number of S-phase and mitotic cells these data indicate that this MAb does not stain normal proliferating cells.
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| C Hydroxyurea |
D Control |
Please find below additional products for a successful use of our Apostain,
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| Catalog Number |
Description |
Clone |
Type |
Quantity. |
Inf. |
Data |
Price |
Quot. |
| AbC156 |
ssDNA (APOSTAIN)
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F7-26 |
Mc |
100 µg |
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Reagents for the preparation and pretreatment Formalin-fixed, paraffin-embedded tissue for in situ hybridizations or immunohistochemistry staining.
AbCys's Proteinase K is a high quality proteinase for pretreatment of formalin-fixed tissue and paraformaldehyde-fixed cells. Each vial contains 5mg of lyophilized powder.
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| Catalog Number |
Description |
Clone |
Type |
Quantity. |
Inf. |
Data |
Price |
Quot. |
| 33801 |
Proteinase K
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HIS |
2 x 5 mg |
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Complementary secondary antibodies:
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Anti-mouse IgM labelled HRPO
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| Catalog Number |
Description |
Clone |
Type |
Quantity. |
Inf. |
Data |
Price |
Quot. |
| LO-MM-3-PO05 |
IgM (mu)
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LO-MM-3 |
Mc |
0,5ml |
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| LO-MM-3-1 |
IgM (mu)
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LO-MM-3 |
Mc |
1ml |
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Anti-mouse IgM labelled FITC
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| Catalog Number |
Description |
Clone |
Type |
Quantity. |
Inf. |
Data |
Price |
Quot. |
| LO-MM-3-F05 |
IgM (mu)
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LO-MM-3 |
Mc |
0,5ml |
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| LO-MM-3-F1 |
IgM (mu)
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LO-MM-3 |
Mc |
1ml |
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DAB Substrate Kit (3,3'-diaminobenzidine)
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| Catalog Number |
Description |
Clone |
Type |
Quantity. |
Inf. |
Data |
Price |
Quot. |
| SK-4100 |
Peroxidase Substrate Kit (DAB)
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Rr |
300 ml after dil. |
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TMB Substrate Kit (3,3', 5,5'-tetramethylbenzidine)
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| Catalog Number |
Description |
Clone |
Type |
Quantity. |
Inf. |
Data |
Price |
Quot. |
| SK-4400 |
Peroxidase Substrate Kit (TMB)
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Rr |
300 ml after dil. |
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4-CN Substrate Kit (4-chloro-1-naphtol)
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| Catalog Number |
Description |
Clone |
Type |
Quantity. |
Inf. |
Data |
Price |
Quot. |
| SK-4300 |
Peroxidase Substrate Kit (4-CN)
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Rr |
300 ml after dil. |
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ABTS Substrate Kit (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)
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| Catalog Number |
Description |
Clone |
Type |
Quantity. |
Inf. |
Data |
Price |
Quot. |
| SK-4500 |
Peroxidase Substrate Kit (ABTS®)
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Rr |
300 ml after dil. |
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Quantitate caspase-mediated apoptosis in whole living
cells without using antibodies
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| Catalog Number |
Description |
Clone |
Type |
Quantity. |
Inf. |
Data |
Price |
Quot. |
| 91 |
FAM FLICATM Poly Caspases Assay Kit (green fluorescent inhibitors)
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Kit |
25 assays |
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| 92 |
FAM FLICATM Poly Caspases Assay Kit (green fluorescent inhibitors)
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Kit |
100 assays |
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| 97 |
FAM FLICATM Caspase 1 Assay Kit (green fluorescent inhibitors)
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Kit |
25 assays |
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| 98 |
FAM FLICATM Caspase 1 Assay Kit (green fluorescent inhibitors)
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Kit |
100 assays |
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| 918 |
FAM FLICATM Caspase 2 Assay Kit (green fluorescent inhibitors)
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Kit |
25 assays |
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| 919 |
FAM FLICATM Caspase 2 Assay Kit (green fluorescent inhibitors)
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Kit |
100 assays |
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| 93 |
FAM FLICATM Caspases 3 & 7 Assay Kit (green fluorescent inhibitors)
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Kit |
25 assays |
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| 94 |
FAM FLICATM Caspases 3 & 7 Assay Kit (green fluorescent inhibitors)
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Kit |
100 assays |
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| 95 |
FAM FLICATM Caspase 6 Assay Kit (green fluorescent inhibitors)
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Kit |
25 assays |
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| 96 |
FAM FLICA Caspase 6 Assay Kit (green fluorescent inhibitors)
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Kit |
100 assays |
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| 99 |
FAM FLICATM Caspase 8 Assay Kit (green fluorescent inhibitors)
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Kit |
25 assays |
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| 910 |
FAM FLICATM Caspase 8 Assay Kit (green fluorescent inhibitors)
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Kit |
100 assays |
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| 912 |
FAM FLICATM Caspase 9 Assay Kit (green fluorescent inhibitors)
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Kit |
25 assays |
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| 913 |
FAM FLICATM Caspase 9 Assay Kit (green fluorescent inhibitors)
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Kit |
100 assays |
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| 922 |
FAM FLICATM Caspase 10 Assay Kit (green fluorescent inhibitors)
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Kit |
25 assays |
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| 923 |
FAM FLICATM Caspase 10 Assay Kit (green fluorescent inhibitors)
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Kit |
100 assays |
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| 929 |
FAM FLICATM Caspase 13 Assay Kit (green fluorescent inhibitors)
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Kit |
25 assays |
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| 930 |
FAM FLICATM Caspase 13 Assay Kit (green fluorescent inhibitors)
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Kit |
100 assays |
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ICT’s Protease Kits :
• Are easy Just add the reagent directly to the cell culture and incubate.
• Are fast The reactions start within 15 minutes of addition to the cells, however we recommend an incubation of 1-4 hours.
• Do not kill the cells The reagents may be incubated up to 72 hours without killing the cells.
• Are brighter Carboxyfluorescein generates a brighter signal than FITC.
• Get into cells better Our reagents are nonpolar, so they pass right thru the cell membrane.
• Use whole living cells You can study apoptosis as it occurs in the cell.
• No lysis or permeabilization steps required You don’t have to destroy the cell to study it.
• Generate better data There is no interference from pro-caspases or inactive forms of the enzyme.
• Do not use antibodies They use substrate and inhibitor peptide sequences linked to a fluorescent label.
• Are more reliable Only cells with active enzymes fluoresce.
• Detect apoptosis earlier than Annexin V and TUNEL assays Caspases are released before the phospholipid turnover & DNA laddering.
• Are flexible Protocols can be customized to fit your experiment.
• Are qualitative and quantitative Read with a fluorescence plate reader, fluorescence microscope, or flow cytometer.
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Figure 2. Jurkat cells dually stained with Hoechst and FAM-VADFMK.
Caspase activity is revealed by green fluorescence in cell #2, indicating that only this cell is apoptotic.
Cell #1 is also dying (scattered blue), but is not apoptotic because it is not green.
Cell #3 is healthy (concentrated blue nucleus).
ICT’s FLICA™ kits measure active caspases 1, 2, 3, 6, 7, 8, 9, 10, 13, or all of them at once.
The FLICA™ reagents (Fluorescent Labeled Inhibitors of Caspases) are comprised of 3 segments - they include: a red (SR = sulforhodamine) or green (FAM = carboxyfluorescein) fluorescent label; an amino acid peptide inhibitor sequence targeted by the active caspase, such as LETD (which is targeted by caspase 8); and a fluoromethylketone group (FMK) which acts as a leaving group and forms a covalent bond with the active enzyme.
The FLICA™reagents are cell permeant, so you don’t have to lyse or permeabilize the membrane to get it into the cell.
Just culture your cells, add the FLICA™ probe to the media, incubate, and wash the cells.
If there are active caspases, they will bind to FLICA™ and retain it within the cell, thereby capturing the fluorescent signal.
The FAM and SR FLICA™ probes constantly fluoresce, therefore any unbound reagent must be washed back out of the cell to remove any background noise. This is done by several quick rinse and spin steps, or further incubation with the wash buffer or media.
The resulting positive fluorescent signal can be detected with a fluorescence microscope (Figures 2, 3, 5, 7, & 9), a fluorescence plate reader (Figure 10), or a
flow cytometer (Figures 1 & 7). The green FLICA™ probe, FAM, excites at 490nm and emits at 520nm. The red FLICA™ probe, SR, excites at 560nm and emits at 600nm.
You can detect caspase activity in individual cells or culture them up to 1x106 cells/mL.
They also work with adherent cells (Figure 3). FLICA™ kits have been used successfully with whole paramecuim, drosophila embryos, thin tissue sections, frozen sections, whole organs, and have even been injected intravenously into small chickens and mice (please call us for details).
To detect apoptosis in general, use the green or red poly caspases kits. These kits use a general peptide sequence (VAD) which reacts with all active caspases. To target a specific caspase, ICT offers several green FAM-FLICA™ kits which react with different caspases (caspase 1, 2, 3&7, 6, 8, 9, 10, or 13) and red SR-FLICA™ kits which react with all caspases, 3&7, and caspase 9. Use the red and green kits together and with our serine protease and cholinesterase kits for duallabeling studies (Figures 7 and 9).
See a list of all green and red FLICA™ kits and pricing on page 14.
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Figure 3. After exposure to kainic acid over time, and labeling with SR-VAD-FMK (kit #916 and #917) for 1 hour, rat brain cortical cells in Figure 3 show increasing apoptosis.
Untreated control cells (3a) exhibit little caspase activity. In this experiment, cells were treated with kainic acid for 40 minutes (3b), 4 hours (3c), and 24 hours (3d) revealing increasing caspase activity and apoptosis.
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Quantitate apoptosis in animals with FLIVOTM
Researchers can now quantify apoptosis in living animals using AbCys's new FLIVO in vivo Fluorescence Apoptosis Detection Kits. Live animal models are invaluable tools for cellular research, particularly in tumor biology and proliferation studies. Unfortunately, the trauma of sacrificing an animal or excising tissue artificially increases the levels of apoptosis in experimental and control animals. This artifact can be eliminated by labeling apoptotic cells in vivo. You will get a true representation of the induction of apoptosis as a result of the treatment without measuring apoptosis resulting from the manipulation of tissues. FLIVO kits are based on FLICA™ reagents(Fluorescent Labeled Inhibitors of Caspases), which measure active caspases inside living cells. The first generation of these kits was successfully used to detect active caspases in whole living cells grown in culture, in whole drosophila embryos, in live paramecia, and even in yeast.
Abcys has now optimized its FLIVO™reagents for injection into live animals.
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Advantages :
Use whole animals Label apoptotic tissues before sacrifice so further manipulation of the tissues won't cause artefacts.
- Live Animals - Use whole animals. Label apoptotic tissues before sacrifice so further manipulation of the tissues won't cause artefacts.
- 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.
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FLIVOTM to monitor the efficacy of chemotherapy.
Picture 1a: mouse window
chamber model
Picture 1b: no killing yet apoptosis
before treatment a very
low level of caspase activity.
Picture 1c: lots of killing apoptosis
after 3 hours of treatment
a very high level of caspase activity
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Small Green FLIVO™ kits contain: FAM-FLIVO™ reagent (1 vial) and injection buffer. Cat.# 980, (enough for 6 rats or mice).
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| Catalog Number |
Description |
Clone |
Type |
Quantity. |
Inf. |
Data |
Price |
Quot. |
| 980 |
Green FLIVO™ In Vivo Apoptosis Detection Kits
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Kit |
6 souris |
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Large Green FLIVO™ kits contain: FAM-FLIVO™ reagent (4 vials) and injection buffer. Cat.# 981, (enough for 25 rats or mice).
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| Catalog Number |
Description |
Clone |
Type |
Quantity. |
Inf. |
Data |
Price |
Quot. |
| 981 |
Green FLIVO™ In Vivo Apoptosis Detection Kits
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Kit |
24 souris |
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Small Red FLIVO™ kits contain: SR-FLIVO™ reagent (1 vial) and injection buffer. Cat.# 982, (enough for 6 rats or mice).
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| Catalog Number |
Description |
Clone |
Type |
Quantity. |
Inf. |
Data |
Price |
Quot. |
| 982 |
Red FLIVO™ In Vivo Apoptosis Detection Kits
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Kit |
6 souris |
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Large Red FLIVO™ kits contain: SR-FLIVO™ reagent (4 vials) and injection buffer. Cat.# 983, (enough for 25 rats or mice).
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| Catalog Number |
Description |
Clone |
Type |
Quantity. |
Inf. |
Data |
Price |
Quot. |
| 983 |
Red FLIVO™ In Vivo Apoptosis Detection Kits
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Kit |
24 souris |
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In Real Time: Monitor Apoptosis with Magic RedTM
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To watch apoptosis develop in real time, use the Magic Red kit (#935,). This reagent uses a substrate peptide sequence of caspases 3&7, so it does not fluoresce until it's actually cleaved by active caspases 3&7. We typically watch color develop over several hours, and have presented data over 16 hours.
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Rat fibroblasts were seeded in 12 well plates at 10,000 cells in 1mL and irradiated the following day. 500uL was removed; 23.1uL of MR-DEVD solution (cat. #935) was mixed with 200uL media and added. 1 hour later, 300uL media was added and cells were photographed over 16 hours. Red fluorescence became brighter as caspase activity and apoptosis progressed. Time 0 is at left, 16 hours is at right. Data courtesy of Dr. Martin Purschke, MA General Hospital.
Caspases 3&7 Apoptosis Magic Red™ Kits
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| Catalog Number |
Description |
Clone |
Type |
Quantity. |
Inf. |
Data |
Price |
Quot. |
| 935 |
Magic RedTM Caspases 3 & 7 Assay Kit (red fluorescent substrates)
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Kit |
25 assays |
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| 936 |
Magic RedTM Caspases 3 & 7 Assay Kit (red fluorescent substrates)
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Kit |
100 assays |
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Votre contact :
Manuel SACHA (Product Manager)
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