Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections.
Since DNA fragmentation is a key feature of programmed cell death (PCD) and also occurs in certain stages of necrosis, we have adapted the methodology of in situ nick-translation (ISNT) to detect DNA fragmentation on a single-cell level. We first established the technique for cell preparations. Apoptosis was induced by gamma-irradiation on freshly isolated rat thymocytes. After fixation procedures, ISNT was performed by overnight incubation either with fluorescein-12-dUTP or with digoxigenin-labeled 11-dUTP and DNA polymerase I. The enzymatic incorporation of labeled nucleotides at sites of DNA fragmentation was detected by flow cytometry either directly or indirectly with fluorescein-conjugated anti-digoxigenin. The quantitative results demonstrated close correlation with morphological essays for apoptosis, DNA gel electrophoresis, and ISNT. Proliferating cells determined by bromodeoxyuridine immunofluorescence were not labeled by ISNT. Immunocytochemistry for cell surface antigens in combination with ISNT allowed the identification of specific cell types undergoing PCD. Furthermore, the simultaneous application of photolabeling techniques with ethidium monoazide and ISNT led to the identification of DNA fragmentation in cells with still intact membranes. Extending ISNT to tissue sections of paraformaldehyde-fixed, paraffin-embedded material reliably revealed labeling of cells with typical morphological features of apoptosis. However, this technique was not useful in detecting early stages of necrotic cell death.
Gold
R
R
Max-Planck-Institute for Psychiatry, Department of Neuroimmunology, Planegg-Martinsried, Germany.
Schmied
M
M
Rothe
G
G
Zischler
H
H
Breitschopf
H
H
Wekerle
H
H
Lassmann
H
H
D000818:Animals; D017209:Apoptosis; D001386:Azides; D002455:Cell Division; D002478:Cells, Cultured; D004249:DNA Damage; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin / Q000031:analogs & derivatives; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000473:pathology; D005434:Flow Cytometry; D005452:Fluoresceins; D005821:Genetic Techniques; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008297:Male; D051381:Rats; D011917:Rats, Inbred Lew; D017207:Rats, Sprague-Dawley; D013950:Thymus Gland / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects
D001386:Azides; D003856:Deoxyuracil Nucleotides; D005452:Fluoresceins; C070202:digoxigenin-11-deoxyuridine triphosphate; C069632:fluorescein-11-deoxyuridine triphosphate; C014535:8-azidoethidium; D004076:Digoxigenin