Optimal prefixation of cells to demonstrate apoptosis by the TUNEL method.
OBJECTIVE
To determine the optimal fixation method for cultured human ovarian cancer cell line SHIN-3 to document cisplatin-induced apoptosis by the terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end-labeling (TUNEL) assay.


METHODS
Cisplatin-treated cancer cell suspensions were (1) fixed in 4% buffered formalin for 10 minutes (BF group); (2) treated with 2% Carbowax in 50% ethanol (CW) for 10 minutes and then fixed in 100% ethanol for one hour (CW + ET); or (3) treated with CW for 10 minutes and then fixed in 4% buffered formalin for one hour (CW + BF). Cell morphology, adhesion to the glass slides and TUNEL reactivity were compared among the three groups. The effects of prolonged prefixation of cell suspensions in CW and of the postfixation of cell smears in BF for one, three and seven days were also examined.


RESULTS
CW + BF treatment yielded satisfactory cell morphology, minimum cell loss and an excellent TUNEL reaction. However, prolonged prefixation in CW resulted in cell shrinkage.


CONCLUSIONS
CW + BF treatment can be widely recommended for use with cytologic preparations for the TUNEL assay.
Kiyozuka
Y
Y
Department of Pathology II, Kansai Medical University, Osaka, Japan. kiyozuka@takii.kmu.ac.jp
Akamatsu
T
T
Singh
Y
Y
Ichiyoshi
H
H
Senzaki
H
H
Tsubura
A
A
D000970:Antineoplastic Agents / Q000627:therapeutic use; D017209:Apoptosis; D019921:Biotinylation; D002945:Cisplatin / Q000627:therapeutic use; D018284:Cystadenocarcinoma, Serous / Q000188:drug therapy / Q000473:pathology*; D053938:DNA Fragmentation; D004253:DNA Nucleotidylexotransferase; D004273:DNA, Neoplasm / Q000302:isolation & purification; D000431:Ethanol; D005260:Female; D006801:Humans; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000473:pathology*; D011092:Polyethylene Glycols; D016707:Tissue Fixation; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D004273:DNA, Neoplasm; D000431:Ethanol; D011092:Polyethylene Glycols; D004253:DNA Nucleotidylexotransferase; D002945:Cisplatin