TUNEL assay
Mouse liver showing an apoptotic cell stained with TUNEL
Terminal deoxynucleotidyl transferase dUTP nickend labeling
(TUNEL) is a method for detecting DNAfragmentation by labeling the
terminal end of nucleicacids.
dUTP
1 MethodTUNEL is a common method for detecting DNA
frag-mentation that results from apoptotic signaling cascades.The
assay relies on the presence of nicks in the DNAwhich can be
identied by terminal deoxynucleotidyltransferase or TdT, an enzyme
that will catalyze the addi-tion of dUTPs that are secondarily
labeled with a marker.It may also label cells that have suered
severe DNAdamage.
2 HistoryOriginally described in the paper by Gavrieli,
Sherman,and Ben-Sasson in 1992,[1] TUNEL has become one ofthe main
methods for detecting apoptotic programmedcell death. However, for
years there has been a debateabout its accuracy, due to problems in
the original as-say which caused necrotic cells to be
inappropriately la-beled as apoptotic.[2] The method has
subsequently beenimproved dramatically and if performed correctly
shouldonly identify cells in the last phase of apoptosis.[3][4]
Newmethods incorporate the dUTPsmodied by uorophoresor haptens,
including biotin or bromine, which can be de-tected directly in the
case of a uorescently-modied nu-cleotide (i.e., uorescein-dUTP), or
indirectly with strep-tavidin or antibodies, if biotin-dUTP or
BrdUTP areused, respectively. Often at late stages of apoptosis,
ad-herent cells are known to detach or pop o. For a reli-able and
reproducible TUNEL imaging assay, the modi-ed nucleotide must not
only be an acceptable substratefor TdT, but the detection method
must also be sensitivewithout bringing about any additional loss of
cells fromthe sample.
3 References[1] Gavrieli Y, Sherman Y, Ben-Sasson SA (1992).
Identication of programmed cell death in situ viaspecic labeling
of nuclear DNA fragmentation. J CellBiol 119 (3): 493501.
doi:10.1083/jcb.119.3.493.PMC 2289665. PMID 1400587.
[2] Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H,Bukowska K,
Bursch W, Schulte-Hermann R (1995). Insitu detection of fragmented
DNA (TUNEL assay) fails todiscriminate among apoptosis, necrosis,
and autolytic celldeath: a cautionary note. Hepatology 21 (5):
14658.doi:10.1002/hep.1840210534. PMID 7737654.
[3] Negoescu A, Lorimier P, Labat-Moleur F, Drouet C,Robert C,
Guillermet C, Brambilla C, Brambilla E(1996). In situ apoptotic
cell labeling by the TUNELmethod: improvement and evaluation on
cell prepa-rations. J Histochem Cytochem 44 (9):
95968.doi:10.1177/44.9.8773561. PMID 8773561.
[4] Negoescu A, Guillermet C, Lorimier P, BrambillaE,
Labat-Moleur F (1998). Importance of DNAfragmentation in apoptosis
with regard to TUNELspecicity. Biomed Pharmacother 52 (6):
2528.doi:10.1016/S0753-3322(98)80010-3. PMID 9755824.
1
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