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.
Sample material: Cells in suspension, cytospin and cell smear preparations, adherent cells grown on slides, and frozen and paraffin-embedded tissue sections.
The In Situ Cell Death Detection Kit, TMR red is based on the detection of single- and double-stranded DNA breaks that occur at the early stages of apoptosis.
Apoptotic cells are fixed and permeabilized. Subsequently, the cells are incubated with the TUNEL reaction mixture that contains TdT and TMR-dUTP. During this incubation period, TdT catalyzes the addition of TMR-dUTP at free 3′-OH groups in single- and double-stranded DNA. After washing, the label incorporated at the damaged sites of the DNA is visualized by flow cytometry and/or fluorescence microscopy.
Kit for the detection and quantification of apoptotic cell death on a single-cell level by flow cytometry and fluorescence microscopy, and for double labeling with fluorescein-labeled cell markers (TMR red).
Precise, fast, and simple technique for detecting and quantitating apoptotic DNA fragmentation at the single-cell level in cells and tissues with a red fluorescent label for fluorescence microscopy and flow cytometry.
In Situ cell death detection kit, TMR red has been used in terminal deoxynucleotidyl transferase biotin–dUTP nick end labeling (TUNEL) assay. It has been used in apoptosis detection assay.
The TUNEL reaction mixture is prepared by mixing the Enzyme Solution and the Label Solution prior to use.
Working concentration: Enzyme concentration
The optimal enzyme concentration range from 0.5 to 5 U per assay. For a standard 50 μl PCR, we recommend using 2 U of the enzyme blend.
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.
Storage conditions (working solution): The TUNEL reaction mixture should be prepared immediately before use and should not be stored. Keep TUNEL reaction mixture on ice until use.