PATHOLOGY & ONCOLOGY RESEARCHVol. 2 No. 2, 1996

 Method

Detection of Drug-induced Apoptosis by Flow Cytometry after Alkaline Extraction of Ethanol Fixed Cells

Rudolf MIHALIK1, Ferenc UHER1, Éva POCSIK1, Lajos BERCZI2, Miklós BENCZUR1, László KOPPER2

1Department of Immunology, National Institute of Hematology, Blood Transfusion and Immunology, Budapest, Hungary
2First Institute of Pathology and Experimental Cancer Research, Semmelweis University of Medicine, Budapest, Hungary

 

A new flow cytometric method was developed to detect apoptotic cells with fragmented DNA and to determine cell cycle distribution of viable cells, in the same sample, by propidium iodide staining. Apoptosis, in HT58 human B lymphoma cells, was induced by etoposide and/or by staurosporine. Using appropriate alkaline solutions (between 1-10 mN NaOH in 150 mM saline) followed by neutralization with buffer solution, the fragmented DNA can be extracted quantitatively from ethanol fixed cells. Further, good resolution of the cell cycle distribution can be obtained in unimpaired cells without RNase treatment. Furthermore, unlike the widely used hypotonic-detergent extraction of unfixed cells, the suggested extraction method can prevent drug-induced disintegration of dead cells when karyorrhexis occurs. Pathology & Oncology Research, Vol 2, Nr 2, 78-83, 1996

Key words: apoptosis; cell cycle; drug-induced; DNA extraction; flow cytometry


Received: Jan 6, 1996; accepted: Feb 2, 1996
Correspondence: László KOPPER, First Institute of Pathology and Experimental Cancer Research, Semmelweis University of Medicine, Üllõi út 26. Budapest H-1085, Hungary; Tel: 36-1-1331142, Fax: 36-1-1331142

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