Terminal deoxynucleotidyl transferase TDT

Fig. 8. Morphological changes of apoptotic eosinophils induced by dexamethasone (Z2). After eosinophils were treated (a) without or (b) with dexamethasone (2 /u,M) for 12 h, cells were harvested and detected by TUNEL assay using the In Situ Cell Death Detection Kit (Boehringer Mannheim). Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by proteinase K and incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT). After washing to remove unbound enzyme conjugated antibody, the horseradish peroxidase retained in the immune complex was visualized by a substrate reaction with diaminobenzidine. The cell nucleus was counterstained with methanol green. Apoptotic eosinophils with nuclear DNA breaks were seen to stain dark brown using a Nikon Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan) in Fig. 8b.

Terminal deoxynucleotidyl transferase (TdT) buffer, 5X concentration 500 mM sodium cacodylate, 10 mM cobalt chloride, 1 mM dithiothreitol Add water to the sodium cacodylate, cool to 0°C on ice, add dithiothreitol, and then cobalt chloride dropwise, stirring continuously. Store in aliquots at —20°C... 

Talbot, David, 190 Tandem dyes, 71-72, 255 Tape back-up, 43 Temporal resolution, 17 Terminal deoxynucleotidyl transferase (TdT), 153... 

Biochemically, apoptosis is characterized by the internucleosomal degradation of chromosomal DNA to form a series of double-stranded fragments that are multiples of 180 200 base pairs in length. These fragments give a characteristic DNA ladder pattern on gel electrophoresis [91, 92] and can be detected by several cytochemical methods, the most extensively used being the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated dUTP nick end labeling (TUNEL) [93-95], The detection of ladder pattern and TUNEL positivity has been adopted as a marker of apoptosis. 

Terminal deoxynucleotidyl transferase (TdT) polymerizes DNA by the same mechanism as DNA polymerases but lacks a requirement for a template DNA strand to direct synthesis (27, 28). TdT prefers single-stranded DNA or duplex DNA containing 3 protruding ends, and is often used in the synthesis of a homopolymeric tail. Nucleotide incorporation is influenced by the divalent cation in the reaction Purine nucleotides are incorporated preferentially in the presence of Mg +, while incorporation of pyrimidine nucleotides is favored in the presence of Co. The use of nucleotide analogs (dideoxynucleotides or cor-dycepin triphosphate) results in the incorporation of a single nucleotide (29). 

TUNEL terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick end-labeling assay... 

Terminal deoxynucleotidyl transferase (TdT) can be purchased from Boehringer Mannheim (Mannheim, Germany) and is supplied as 25 units/pL and stored at -20°C. 

Chapman, R. S., Chresta, C. M Herberg, A. A., Beere, H. M., Heer, S., Whetton, A. D Hickman, J. A., and Dive, C. (1995) Further characterisation of the in situ terminal deoxynucleotidyl transferase (TdT) assay for the flow cytometric analysis of apoptosis in drag resistant and drug sensitive leukaemic cells. Cytometry 20,245-256. 

Standard 40 pL reaction mix 1 pL scissile strand oligonucleotide (approximately 10 picomoles) with 20 pL purified distilled water, 8 pL 5X labeling buffer, 10 pL [a32P]-cordycepin and 1 pL terminal deoxynucleotidyl transferase (TdT) (15 units). 

Mathewson RC, Kjeldsberg CR, Perkins SL. Detection of terminal deoxynucleotidyl transferase (TdT) in non-hematopoietic small round cell tumors of children. Pediatr Pathol Lab Med. 1997 17 835-844. 

Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase active during the process of immunoglobulin and T-cell receptor gene rearrangement early in a precursor B or T cell s life. Only normal early B and T lymphoblasts mark for TdT. The staining pattern is nuclear. TdT is a sensitive and specific antibody for lymphoblastic lymphoma/leukemia because only a small proportion of myeloid leukemia cases are positive (Fig. 6.13). 

Lymphoblastic lymphoma and acute lymphoblastic leukemia are morphologically and immunophenotypi-cally the same disease and are distinguished on clinical grounds. Although the majority of lymphoblastic leukemias are of B lineage, only approximately 20% of lymphoblastic lymphomas express B-cell markers. Practically all cases of lymphoblastic leukemia/lym-phoma produce an enzyme, terminal deoxynucleotidyl transferase (TdT), involved in gene rearrangement. TdT marks the nucleus of lymphoblasts. CD 19 is expressed in almost all lymphoblastic lymphomas but... 

Other members of this family include eukaryotic terminal deoxynucleotidyl transferase (TdT), Pol /i, Pol A, and Pol a. TdT is known to function in antigen receptor diversification during V(D)J recombination through addition of nucleotides (N additions) to gene segment junctions in a template-independent manner (reviewed in Fowler and Suo ). The more recently discovered Pol A and Pol /t have been implicated in... 

Terminal deoxynucleotidyl transferase (TdT), an enzyme found in bone marrow and thymus tissue, can extend a DNA primer by 5 —> 3 polymerization using deoxyri-bonucleoside triphosphates as substrates. The primer must be at least three nucleotides... 

Mammalian cells contain a template-independent family X polymerase called terminal deoxynucleotidyl transferase (TdT). TdT contains a... 

Artemis-DNA-PKcs acts as a nuclease, trimming the ends and increasing the diversity at the junctions (Ma et al, 2002). At this intermediate step, terminal deoxynucleotidyl transferase (TdT) (Gilfillan et al, 1993 Kohler and Milstein, 1975 Komori et al, 1993), can add extra nucleotides... 

Terminal deoxynucleotidyl transferase (TdT) 15U/pL and 5X reaction buffer 0.5M potassium cacodylate (pH 7.2), lOtnM cobalt chloride, 1 mAf DTT (Gibco-BRL 18008-011). 

Terminal deoxynucleotidyl transferase (TdT, 10-20 U/pL) can be purchased from Gibco-BRL. The enzyme is supplied with a vial of 5X reaction buffer (500 mM potassium cacodylate, pH 7 2, 10 mM C0CI2,1 mM DTT) Store at -20°C. Note that potassium cacodylate and C0CI2 are highly toxic and should be handled with care... 

Another common method for measuring apoptosis is the DNA labeling technique, terminal deoxytransferase-mediated dUTP nick-end labeling (TUNEL). In this method, labeling of DNA strand breaks at the 3 end is achieved by using the enzyme, terminal deoxynucleotidyl transferase (TdT) and either a radioactive or a fluorescently-Iabeled deoxynucleotide. This method provides a very sensitive measurement of DNA strands breaks. However, as DNA strand... 

Intracellular proteins associated terminal deoxynucleotidyl-transferase (TdT) Flow-cytometry based bead assay nick end labeling of broken ends of the double stranded DNA Detects proteins from cells associated with different stages of Fluorescence... 

The same group also showed how to control the size of amphiphilic DNA block copolymer nanoparticles by enzymatic reaction. In brief, particles obtained from block copolymers synthesized in a DNA synthesizer were incubated with terminal deoxynucleotidyl transferase (TdT) at 37°C in the presence of Co " and... 

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