Monoclonal antibody 2E12 was prepared by immunization of mice with cells of a chronic myeloid leukemia cell line MOLM-7. Human hematopoietic cell lines JURKAT, HPB-ALL, RC2A and MOLM-7 were induced to receptor mediated apoptosis by the treatment with anti-Fas monoclonal antibody 7C11 and subsequently tested for reactivity with 2E12 antibody in comparison to staining with annexin V-FITC and PI in the two-color immunofluorescence and flow cytometry. After 2, 5, 24, and 48 hours of induction, a gradual increase of the percentage of 2E12 positive cells in all cell lines was observed, which partially correlated with an increase of annexin V-FITC binding with a delay of about 12 hours. In the two- color fluorescence microscopy the 2E12 antibody positivity was restricted to the annexin V positive cells, but their number was lower. The binding of 2E12 did not induce apoptosis nor influenced the binding of annexin V. We suppose that the antibody 2E12 detects an antigen expressed on a subpopulation of cells in death. Therefore it can be useful as a new marker for further dissection between living, apoptotic and necrotic cellular populations in vitro.

Department of Cellular Biochemistry Institute of Hematology and Blood Transfusion Prague 2 12820 Czech Republic

Interaction of late apoptotic and necrotic cells with vitronectin