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11684795910 Roche

In Situ Cell Death Detection Kit, Fluorescein

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Properties

Quality Level   100
usage   sufficient for ≤50 tests
mfr. no.   Roche
shipped in   dry ice
storage temp.   −20°C

Description

General description

Kit for the detection and quantification of apoptosis at the single-cell level, based on labeling of DNA strand breaks (TUNEL technology); analysis by fluorescence microscopy or flow cytometry.

Widely used methods to determine apoptosis include the analysis of the genomic DNA by agarose-gel electrophoresis and DNA fragmentation assays based on 3H-thymidine and, alternatively, 5-Bromo-2′-deoxy-uridine. The methods involve the separation of fragmented, low molecular weight DNA from unfragmented, high molecular weight DNA in a given cell population. Thus, these methods do not provide information about the fate of an individual cell in a given cell population, or particularly, in tissue sections. Alternatively, individual apoptotic cells may be microscopically recognized because of the characteristic appearance of nuclear chromatin condensation and fragmentation, but this method is subjective and limited to a relatively narrow time window when the morphological changes are at a maximum.
The hallmark of apoptosis is DNA degradation, which in early stages, is selective to the internucleosomal DNA linker regions. The DNA cleavage may yield double-stranded and single-stranded DNA breaks (nicks). Both types of breaks can be detected by labeling the free 3′-OH termini with modified nucleotides (e.g., biotin-dUTP, DIG-dUTP, fluorescein-dUTP) in an enzymatic reaction. The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes the template-independent polymerization of deoxyribonucleotides to the 3′-end of single- and double-stranded DNA. This method has also been termed TUNEL (TdT-mediated dUTP-X nick end labeling). Alternatively, free 3′-OH groups may be labeled using DNA polymerases by the template-dependent mechanism called nick translation. However, the TUNEL method is considered to be more sensitive and faster.

Specificity

The TUNEL reaction preferentially labels DNA strand breaks generated during apoptosis. This allows discrimination of apoptosis from necrosis and from primary DNA strand breaks induced by cytostatic drugs or irradiation.

Application

The In Situ Cell Death Detection Kit, Fluorescein,  is a precise, fast, and simple nonradioactive technique to detect and quantify apoptotic cell death at the single-cell level by fluorescence microscopy and quantitative detection by flow cytometry in cells and tissues. Thus, the In Situ Cell Death Detection Kit can be used in many different assay systems.
Examples are:
• Detection of individual apoptotic cells in frozen and formalin-fixed tissue sections in basic research
• Determination of sensitivity of malignant cells to drug-induced apoptosis in cancer research
• Typing of cells undergoing cell death in heterogeneous populations by double staining procedures

Features and Benefits

• Sensitive: The direct labeling procedure using fluorescein-dUTP reduces background labeling
• Fast: The use of fluorescein-dUTP allows analysis of the samples directly after the TUNEL reaction
• Convenient: No secondary detection system required
• Accurate: Identification of apoptosis at a molecular level (DNA-strand breaks) and identification of cells at the very early stages of apoptosis

Packaging

1 kit containing 2 components.

Preparation Note

Working solution: Add total volume (50 μl) of Enzyme Solution to the remaining 450 μl Label Solution to obtain 500 μl TUNEL reaction mixture.
Mix well to equilibrate components.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Kit component only

Description

Product #

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Enzyme Solution (TdT)    
Label Solution (fluorescein-dUTP)    
Safety & Documentation

Safety Information

RIDADR 
UN 1556 6.1 / PGIII

Documents

Certificate of Analysis (COA)

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Protocols & Articles

Articles

Cellular Apoptosis Assays | Programmed Cell Death

Apoptosis, or programmed cell death, is a growth-limiting regulatory mechanism by which cells can trigger their own death in response to extracellular signals because of irreparable cellular or DNA d...
Keywords: Apoptosis, Autophagy, Cancer, Cell culture, Detection methods, Diseases, Enzyme-linked immunosorbent assay, Flow cytometry, Immunofluorescence, Immunohistochemistry, Microscopy, Phosphorylations

Protocols

In Situ Cell Death Detection Kit, Fluorescein Protocol & Troubleshooting

Dehydrated fixed tissue sections (2 min dehydration in absolute ethanol, storage at -15 to -25 °C) can be placed directly into the permeabilization solution. They can also first be rehydrated in PBS....
Keywords: Apoptosis, Dehydration reaction, Polymerization reactions, Reductions

Peer-Reviewed Papers
15

References

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