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APT403

Sigma-Aldrich

CaspaTag Caspase 3,7 In Situ Assay Kit, Fluorescein

The In Situ Caspase Detection Kit for Flow Cytometry use a novel approach to detect active caspases. The methodology is based on Fluorochrome Inhibitors of Caspases (FLICA).

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eCl@ss:
32161000

Quality Level

species reactivity (predicted by homology)

mammals

manufacturer/tradename

CaspaTag
Chemicon®

technique(s)

activity assay: suitable
flow cytometry: suitable

NCBI accession no.

UniProt accession no.

detection method

fluorometric

shipped in

wet ice

Gene Information

human ... CASP3(836)

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APT423APT131APT408
species reactivity (predicted by homology)

mammals

species reactivity (predicted by homology)

mammals

species reactivity (predicted by homology)

mammals

species reactivity (predicted by homology)

-

Quality Level

100

Quality Level

100

Quality Level

100

Quality Level

100

manufacturer/tradename

CaspaTag, Chemicon®

manufacturer/tradename

CaspaTag, Chemicon®

manufacturer/tradename

Chemicon®

manufacturer/tradename

CaspaTag, Chemicon®

technique(s)

activity assay: suitable, flow cytometry: suitable

technique(s)

activity assay: suitable, flow cytometry: suitable

technique(s)

activity assay: suitable

technique(s)

activity assay: suitable, flow cytometry: suitable

detection method

fluorometric

detection method

fluorometric

detection method

colorimetric

detection method

fluorometric

General description

Apoptosis is an evolutionarily conserved form of cell suicide, which follows a specialized cellular process. The central component of this process is a cascade of proteolytic enzymes called caspases. These enzymes participate in a series of reactions that are triggered in response to pro-apoptotic signals and result in the cleavage of protein substrates, causing the disassembly of the cell (Slee et al. 1999).

Caspases have been identified in organisms ranging from C. elegans to humans. The mammalian caspases play distinct roles in apoptosis and inflammation. In apoptosis, caspases are responsible for proteolytic cleavages that lead to cell disassembly (effector caspases), and are involved in upstream regulatory events (initiator caspases). An active caspase consists of two large and two small subunits that form two heterodimers which associate in a tetramer (Walker et al. 1994; Wilson et al. 1994; Rotonda et al. 1996). In common with other proteases, caspases are synthesized as precursors that undergo proteolytic maturation, either autocatalytically or in a cascade by enzymes with similar specificity (Kumar 1999).

Caspase enzymes specifically recognize a 4 or 5 amino acid sequence on the target substrate which necessarily includes an aspartic acid residue. This residue is the target for cleavage, which occurs at the carbonyl end of the aspartic acid residue (Thornberry et al. 1997). Caspases can be detected via immunoprecipitation, immunoblotting techniques using caspase specific antibodies, or by employing fluorochrome substrates which become fluorescent upon cleavage by the caspase.

Test Principle:

CHEMICON′s In Situ Caspase Detection Kits use a novel approach to detect active caspases. The methodology is based on Fluorochrome Inhibitors of Caspases (FLICA). The inhibitors are cell permeable and non-cytotoxic. Once inside the cell, the inhibitor binds covalently to the active caspase (Ekert et al. 1999). This kit uses a carboxyfluorescein-labeled fluoromethyl ketone peptide inhibitor of caspase-3 (FAM-DEVD-FMK), which produces a green fluorescence. When added to a population of cells, the FAM-DEVD-FMK probe enters each cell and covalently binds to a reactive cysteine residue that resides on the large subunit of the active caspase heterodimer, thereby inhibiting further enzymatic activity. The bound labeled reagent is retained within the cell, while any unbound reagent will diffuse out of the cell and is washed away. The green fluorescent signal is a direct measure of the amount of active caspase-3 or caspase-7 present in the cell at the time the reagent was added. Cells that contain the bound labeled reagent can be analyzed by 96-well plate-based fluorometry, fluorescence microscopy, or flow cytometry.

Application:

The CHEMICON In Situ FLICA Caspase-3/7 Detection Kit is a fluorescent-based assay for detection of active caspase-3 or caspase-7 in cells undergoing apoptosis. The kit is for research use only. Not for use in diagnostic or therapeutic procedures.

Application

Research Category
Apoptosis & Cancer
The In Situ Caspase Detection Kit for Flow Cytometry use a novel approach to detect active caspases. The methodology is based on Fluorochrome Inhibitors of Caspases (FLICA).

Components

FLICA Reagent (FAM-DEVD-FMK): Four lyophilized vials

10X Wash Buffer: 60 mL

Fixative: 6 mL

Propidium Iodide: 1 mL at 250 μg/mL, ready-to-use

Hoechst Stain: 1 mL at 200 μg/mL, ready-to-use

Storage and Stability

· Store unopened kit materials at 2-8°C up to their expiration date.

· Reconstituted FLICA Reagent (150X) should be frozen at -20ºC for up to 6 months, and may be thawed twice during this time. Aliquot into separate amber tubes if desired. Protect from light at all times.

· Store diluted (1X) wash buffer up to -20ºC for 2 weeks.

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

pictograms

Skull and crossbonesHealth hazard

signalword

Danger

Hazard Classifications

Acute Tox. 3 Inhalation - Acute Tox. 4 Dermal - Acute Tox. 4 Oral - Carc. 1B - Eye Irrit. 2 - Muta. 2 - Skin Irrit. 2 - Skin Sens. 1 - STOT SE 2 - STOT SE 3

target_organs

Respiratory system

Storage Class

6.1C - Combustible, acute toxic Cat.3 / toxic compounds or compounds which causing chronic effects

wgk_germany

WGK 3


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Bo Hua Hu et al.
Journal of neuroscience research, 88(8), 1812-1821 (2010-01-22)
Acoustic overstimulation causes apoptotic cell death in the cochlea. This death process is mediated, in part, by the mitochondrial signaling pathway involving Bcl-2 family proteins. Myeloid cell leukemia sequence 1 (Mcl-l) is an antiapoptotic member of the Bcl-2 family. Its
Qunfeng Cai et al.
Neurobiology of disease, 45(2), 723-732 (2011-11-03)
Cell-cell junctions and junctions between cells and extracellular matrix are essential for maintenance of the structural and functional integrity of the cochlea, and are also a major target of acoustic trauma. While morphological assessments have revealed adhesion dysfunction in noise-traumatized
Mohammad Husein Abnosi et al.
Iranian journal of basic medical sciences, 15(4), 900-906 (2013-03-16)
Arsenic compounds are potent human carcinogen and produce a variety of stress responses in mammalian cells. Recently sodium arsenite has been recommended to be used as anti malignancy drug by American food and drug administration (FDA). In this study, we
Lisa L Cunningham et al.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 22(19), 8532-8540 (2002-09-28)
Aminoglycoside exposure results in the apoptotic destruction of auditory and vestibular hair cells. This ototoxic hair cell death is prevented by broad-spectrum caspase inhibition. We have used in situ substrate detection, immunohistochemistry, and specific caspase inhibitors to determine which caspases
Golnaz Pakravan et al.
Cell death & disease, 9(7), 758-758 (2018-07-11)
Doxorubicin (Dox) is a widely used powerful chemotherapeutic component for cancer treatment. However, its clinical application has been hampered due to doxorubicin-induced cardiomyopathy upon the cessation of chemotherapy. Previous studies revealed that PPARγ plays a crucial protective role in cardiomyocytes.

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