CASPASE 3 7 APOPTOSIS CASPASE DETECTION KITS FOR IN VITRO USE IN REAL TIME MAGIC RED

Caspase 3&7 Apoptosis Detection Kits
Detect caspase activity in whole living cells (green and red fluorescence).
Monitor caspase activity in real time (red fluorescence).

Normal (left) and keratoconus (right) corneal fibroblasts were labeled with FAM-FLICA™ Caspases 3&7 (kit cat.# 93, see below).

Magic Red Caspases 3&7 kit, Cat. #935

ICT has 3 test kits to quantify and monitor caspases 3 & 7:
     1. in vitro detection with green fluorescence (FAM-FLICA™)
     2. in vitro detection with red fluorescence (SR-FLICA™)
     3. real-time monitoring with red fluorescence (Magic Red™)

  These kits are not ELISAs and do not use antibodies. Instead, they use a substrate or inhibitor sequence targeted by  active caspases linked to a green or red fluorescent probe. The reagents are cell-permeant, so you don't have to lyse the cells or permeabilize the membrane - just add the reagent to the media. If there are active caspases inside the cell, they will either:

     1. Bind to the inhibitor sequence, in the case of ICT's green and red FLICA™ kits.
     2. Cleave the substrate sequence, in the case of ICT's Magic Red™ red kit.

     These kits are very specific for active caspases. Because these kits require the caspases to bind to an inhibitor or cleave a substrate, they must be activated, so you won't get any reactions from pro-caspases nor inactive forms of the caspases. With all these kits, you can see a clear distinction between apoptotic and necrotic cells. These assays are easier and more reliable than Tunel or Annexin.

Comparison of ICT's 3 different kits

Frequently Asked Questions

References (pdf)

Order now!  1-800-829-3194.

Green (FAM-FLICA™) & Red (SR-FLICA™)

Normal (left) and keratoconus (right) corneal fibroblasts were treated with 200uM H2O2 and labeled with FAM-FLICA™ Caspases 3&7 (kit cat.# 93). Treated keratoconus corneal fibroblasts (right) show a significant increase in caspases 3&7 activity compared to treated normal cells (left). Non-apoptotic cells are dark in background.  Data courtesy of Dr. Cristina Kenney, M.D., Ph.D. Dept. of Ophthalmology, UC Irvine.  Reference: Chwa, M., Atilano, S. R., Reddy, V., Jordan, N., Kim, D. W., Kenney, M. C. 2006. Increased stress-induced generation of reactive oxygen species and apoptosis in human keratoconus fibroblasts. Investigative Ophthalmology and Visual Science May 2006 vol 47, no. 5, 1902-1910.

See our FLICA™ protocols page for tips

References

     The Caspases 3&7 FLICA™ kits are based on an inhibitor peptide sequence (DEVD) targeted by both caspase 3 and by caspase 7.  This sequence is linked to a green or red fluorescent probe. The green probe is carboxyfluorescein (FAM), which is brighter and gets into the cell better than FITC, but uses the same wavelength settings as FITC. The red probe is sulforhodamine (SR). These probes are also linked to a fluoromethyl ketone group (FMK), which helps the active caspase to bind to FLICA™ (Fluorescent-Labeled Inhibitors of CAspases). 
     Because of the non-polar nature and relatively small molecular weight of these FLICA™ probes, they are cell-permeant.  Because FLICA™ automatically crosses the cell membrane, there is no need for any lysis nor membrane permeabilization steps in this assay. It's easy - just add FLICA™ to the media, and it will enter the cell (let the reagent incubate with the cells between 1-4 hours). If there are active caspases, they will form a covalent bond with DEVD. Once bound, the reagent is simply too big to leave the cell and the green or red fluorescent signal is retained within the cell. This binding also prevents the caspase from further catalysis (FLICA™ reagents are inhibitors), but will not stop apoptosis from proceeding. The reagent will start to react with active caspases 3&7 within 15 minutes of addition to the media.
     FLICA™ reagents continually fluorescence, whether bound to a caspase or not. Therefore, a simple wash and spin step must be done after the reagent incubation period to remove any unbound reagent (there may be some background noise from unbound reagent, but this is very low). FLICA™ is so sensitive that it will even detect innate apoptosis in non-induced cells, which is typically 2-6% of all cells.  FLICA™ is specific for active caspases - it will not react with pro-caspases nor inactive forms of the enzyme. Cells can be fixed or paraffin-embedded after labeling with FLICA™. Green FAM- FLICA™ excites at around 490nm and emits at 530nm; red SR- FLICA™ excites at 560 and emits at 590. Read cells with a fluorescence microscope, plate reader, or flow cytometer.
     The green Caspases 3&7 FLICA™ kit uses the reagent FAM-DEVD-FMK and is available in 2 sizes, 25 and 100 tests, catalog # 93 and 94, respectively.
     The red Caspases 3&7 FLICA™ kit uses the reagent SR-DEVD-FMK and is available in 2 sizes, 25 and 100 tests, catalog # 931 and 932, respectively.

See our main FLICA™ page for details

Sample protocol for suspension cells (read manual):
See our FLICA™ protocols page for experiments and data
1. Culture your cells up to 1 x 10⁶ cells/mL. 
2. Induce apoptosis following your protocol, and create positive and negative controls. 
3. Reconstitute the reagent with 50uL DMSO to form the stock concentrate (which can be frozen for future use). 
4. Dilute the stock concentrate with 200uL 1X PBS to form the working solution. 
5. Add ~10uL of the working solution directly to a 300-500uL aliquot of your cell culture for labeling. You may need to use more or less reagent than we suggest.
6. Incubate 1-4 hours. The reagent should start to react with the caspases within 15 minutes.
7. Spin cells ~5 minutes at 220g and carefully remove the FLICA™-labeled supernatant.
8. Add at least 400uL of the FLICA™ 1x apoptosis wash buffer (or use cell culture media).
9. Let the buffer incubate with the cells for 5-15 minutes protected from light (cells may be placed in the incubator).  
10. Carefully aspirate the buffer and discard.
11. Wash the cells up to 3 times by repeating Steps 7-10.  After the final wash, resuspend the cells in 1x apoptosis wash buffer for analysis. Some cells may need to be washed more than others depending on the type of instrument used.  Cells evaluated by flow cytometry may not need to be washed as much as cells evaluated in a plate reader or microscope, as the sheathing fluid acts as a wash buffer.  Cells analyzed in a plate reader often have to be washed the most, as the fluorometer measures total fluorescence within the well and any excess reagent will lead to higher RFUs. 
12. If desired, label cells with Hoechst stain.
13. If desired, label cells with Propidium Iodide or 7AAD.
14. If desired, fix cells. 
15. Analyze data using a fluorescence microscope, plate reader, or flow cytometer.

References

Order now!  1-800-829-3194.

FLICA™ is
easy
fast
reliable
sensitive
quantitative

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Require wash step.

Good for end-point experiments.

Read with fluorescence microscope, plate reader, or flow cytometer.  

FAM-FLICA™ (green) excites at 490 and emits at 520 nm.

FAM-FLICA™ is compatible with DAPI, SR-FLICA, Hoechst, PI, 7-AAD, and other fluorescent reagents for dual-staining studies.

SR-FLICA™ (red) excites at 560 and emits at 600 nm.

SR FLICA™ is compatible with DAPI, FAM-FLICA, Hoechst, 7-AAD, GFP, and other fluorescent reagents for dual-staining studies.

Real Time Kits with Magic Red™

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. Bright field image shows increasing red intensity 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. Watch the color increase in the movie clip of these cells in a bright field.     

ICT's MR Caspases 3&7 kit clearly distinguishes apoptotic MCF-7 cells from non-apoptotic cells. Apoptotic cells exhibit red lysosomes (MR) with less intense blue nuclei. These cells are intermixed with non-apoptotic cells bearing bright blue nuclei (Hoechst) and absent lysosomal staining. Read this publication

MR-Caspases 3&7 Apoptosis Manual

Caspases 3&7 Apoptosis Magic Red™ kits contain: MR-DEVD reagent,
Hoechst 33342, and acridine orange. 

Magic Red Caspases 3&7 kit, Cat. #935

References

     The Caspases 3&7 Magic Red kit is based on a dipeptide substrate sequence (DEVD)₂ linked to a red fluoroscent leaving group, cresyl violet (Magic Red™).  This peptide sequence is cleaved by both active caspase 3 and caspase 7, but does not prevent the enzyme from further catalysis (the MR reagent is a substrate). There is no need to lyse the cells or permeabilize the membrane. 
     MR is a very easy assay - no processing is required. Just add MR to the media and watch the color develop. In fact, we discourage additional manipulation of the cells as the MR fluorophore may migrate back out of the cell.  The reagent will start to react with active caspases 3&7 within 15 minutes of addition to the media.  If there are active caspase 3&7 enzymes, the reagent will fluoresce red; if not, it will not fluoresce.  Watch this happen in 2 movies of real-time caspase activity, courtesy of Dr. M. Purschke, MA General Hospital: movie clip of live cells (bright field) ; movie of red fluorescence only. In typical experiments, the reagent incubates with the cells between 1-4 hours or more.
     In contrast to FLICA™, there is no need to wash out any unbound reagent. Since Magic Red™ will not fluoresce until it is actually cleaved by an active caspase 3 or 7 enzyme, you can actually watch the fluorescence develop over time. As caspase 3&7 activity increases, positive cells will start to fluoresce red. Cells can be fixed or paraffin-embedded after labeling with Magic Red™. MR excites at 540-590 and emits at >610nm. This signal can be detected with a fluorescence microscope, a fluorescence plate reader, or special flow cytometers with adjustable excitation wavelengths.
     Caspases 3&7 are active in the cytosol. After staining with MR, there are often areas of bright red fluorescence located within lysosomal structures. However, caspases are not known to be present within lysosomes.  The presence of the red fluorescent product within the lysosomes is most likely due to the migration and concentration of the cleaved red fluorescent fluorophore from the cytosol into the lysosomes, thereby creating bright spots.
     The red Caspases 3&7 Magic Red kit uses the reagent MR-(DEVD)₂ and is available in 2 sizes, 25 and 100 tests, catalog # 935 and 936, respectively.

See our main Magic Red™ page for details

Sample Protocol (manual)
1. Culture your cells individually or up to 1 x 10⁶ cells/mL. 
2. Induce your experimental conditions following your protocol. 
3. Reconstitute the reagent with DMSO to form the stock concentrate (which can be frozen for future use). 
4. Dilute the stock concentrate with 1X PBS to form the working solution. 
5. Add ~10uL of the working solution directly to a 300-500uL aliquot of your cell culture for labeling. 
6. Incubate, typically 1-4 hours; protect cells from light.
7. If desired, label DNA with Hoechst stain. 
8. If desired, label lysosomes with Acridine Orange. 
9. If desired, fix cells. 
10. Analyze data using a fluorescence microscope, or plate reader.

Reference
Lee, B., G. L. Johnson, S. A. Hed, Z. Darzynkiewicz, J. Talhouk, and S. Mehrotra.  2001.  DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)₂-cresyl violet.  Biotechniques vol 35 no 5:1080-1085. Read this publication . 

Read all references (pdf).

Order now!  1-800-829-3194.

MR kits are
easy
fast
reliable
sensitive.

Try it today
1-800-829-3194
Order

Dual staining of apoptotic MCF-7 cells reveals areas of general caspase activity, indicated by green fluorescence from FAM-FLICA™ Poly-Caspases Kit (cat. # 92). Red fluorescence generated from MR-Caspases 3&7 (cat. # 936) indicates areas of caspase 7 activity (MCF-7 cells are deficient in caspase 3).

Substrate-based

No end-point, can monitor cells for many hours

No wash step

Fluorophore accumulates in lysosomes and may exit the cell as the membrane becomes porous.

Read with fluorescence microscope or plate reader. 

MR excites at 540-590 and emits at >610 nm.

Frequently Asked Questions

How specific are your reagents for each caspase? 
They will initially react with the target caspase – the reagents have a preference for a certain caspase. However, over time the reagents will become less specific. The longer the reagent incubates in the cell, the more they will start to bind to other caspases and enzymes. We suggest incubation periods of 15 minutes to 1 hour; however many researchers incubate for 3 hours and longer. Our reagents FAM-DEVD-FMK, SR-DEVD-FMK, and MR-(DEVD)₂ react with both caspase 3 and with caspase 7. We have found that some non-caspase enzymes may eventually contribute to a low level of signal. Include a negative control in your experiment to determine a baseline level of apoptosis occurring in your cell population. Also include a positive control in your experiment to determine what the high level of apoptosis could be (apoptosis can be induced using staurosporine or camptothecin, see ICT's Accessory Reagents). Compare the baseline fluorescence with your samples and with the high control level of fluorescence to ensure that your experiment is working.

What is an inhibitor? 
An inhibitor is an amino acid sequence that when bound by the catalytic site of the enzyme prevents the enzyme from further catalysis. ICT’s FLICA reagents are based on inhibitors and use caspase-targeting amino acid sequences which were previously characterized by using cold (unlabeled) caspase inhibitors such as z-DEVD-FMK. The caspases 3&7 kits use the inhibitor sequence DEVD. This is linked to a green or red fluorescent label, FAM (carboxyfluorescein = green) or SR (sulforhodamine = red), and also to a carboxy terminal FMK reactive group = fluoromethyl ketone.  The green FLICA reagent is often noted as FAM-DEVD-FMK, and the red FLICA reagent is often noted as SR-DEVD-FMK

What is a substrate? 
A substrate is an amino acid sequence that is cleaved by the catalytic site of the enzyme. Substrates do not prevent the enzyme from further catalysis. ICT’s Magic Red kits use substrate sequences linked to a fluorphore. ICT’s Caspases 3&7 kit uses the sequence (DEV)₂ linked to the Magic Red fluorophore, cresyl violet. When linked together, the fluorophore is quenched. However, once cleaved by active caspase 3 and 7 enzymes, the red fluorescent signal from cresyl violet is detectable.

My non-stimulated controls also generate a signal – what’s going on? 
Background apoptosis ranges from 2-8% depending on the cell line and your growing conditions.  You are always going to see a certain level of apoptosis. The non-stimulated controls should also have a low percentage of apoptotic cells. With the FAM and SR FLICA kits, there may be a small amount of background fluorescence if some of the unbound reagent is not completely washed out of the cell. The MR kit may generate a background signal if the cleaved red fluorophore leaves the cell (MR may also concentrate inside lysosomes, forming bright spots).

How long does the assay take?  
You can start and finish your experiment in one day. You may even be able to finish in 1-2 hours, depending upon the reactivity of your particular cell line. FLICA™ and MR will start to react with caspases as soon as it is added to the media. Color may be visualized in positive cells within 15 minutes of addition to the cells. Typical reagent incubation periods for FLICA™ are 1-4 hours; MR can be incubated indefinitely during the monitoring procedure. We have incubated cells with FLICA and MR for 72 hours without killing the cells. Adherent cells can be trypsinized before or after labeling. The exact incubation time should be determined by your experimental protocol.  

What is “1 test”? 
To standardize the manual, “1 test” is a 300uL aliquot of suspension cells grown up to 1x10^6 (maximum) for testing on a fluorescence plate reader. We typically analyze 200,000 cells/well in a plate reader. Cells can be trypsinized and run through a flow cytometer. MR also works with just a few cells for viewing under a fluorescence microscope.

How many cells do I need?
You need 300uL of cells should be grown to a maximum of 1x10⁶ cells/mL, beyond that, they will begin to naturally enter apoptosis due to over-crowding. Generally between 0.5x10⁵ to 5x10⁵ cells/96-well are needed.

How much reagent do I use? 
The reagents are provided as stabilized lyophilized powders. The vial may appear empty, but it's not. The reagent will appear as an iridescent sheen inside the vial. Reconstitute FLICA™ ("25 tests/vial") with 50uL DMSO; further dilute with 200uL PBS. Reconstitute the small Magic Red™ "25 test" vial with 50uL DMSO; reconstitute the large Magic Red™ "100 test" vial with 200uL DMSO; dilute 1:5 with PBS. Add ~10uL of the working solutions to your cells. Your particular cells may require more or less reagent than we suggest. The first time you use the kit, set up a titration experiment to determine the optimal amount of reagent to add to your cells. Protect the reagent and treated cells from light as the reagent will photobleach over time. 

Will it work with fixed or paraffin or frozen cells?
FLICA™ and Magic Red™ kits will not work on fixed cells (but you can fix the cells after labeling), nor will they work with paraffin embedded cells. The reagents require the enzymes to be active, and fixation and paraffin inactivates the enzymes. FLICA™ works with suspension cells, thin frozen tissues, adherent cells, and primary neurons grown on a feeder layer. Magic Red™ kits may work with suspension cells, adherent cells, some frozen cells and thin frozen sections; Magic Red™ will not work with cells grown on a feeder layer - too much overall backgrounds. FLICA™ and Magic Red™ reagents require the cell membrane to remain intact to contain the fluorescent probe within the cell. Freezing often creates pores in the membranes, allowing the positive fluorescent signal to leave the cell. If this is a problem, all the cells may appear stained as the probe travels throughout the sample.

Order now!  1-800-829-3194.

References

Magic Red 3&7 Kits:
1. Lee, B. W., Johnson, G. L., Hed, S. A., Darzynkiewicz, Z., Talhouk, J., Mehrotra, S. 2003. DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet. BioTechniques vol. 35, no. 5: 1080-1085.

See all other references (pdf)

Please contact Sally Hed with questions 952-888-8788 x10 sally@immunochemistry.com .