02 BOOK OF ABSTRACTS .indd

Quantification of lysosomal fusion in electrofused
hybridoma cells
Mateja Gabrijel 1 , Marko Kreft 1,2 and Robert Zorec 1,2
1
Celica Biomedical Sciences Center, Stegne 21, 1000 Ljubljana, Slovenia; 2 Laboratory
of Neuroendocrinology – Molecular Cell Physiology, Institute of Pathophysiology,
Medical School, University of Ljubljana, 1000 Ljubljana, Slovenia
To enhance the immune response, hybridomas between dendritic and tumor cells,
representing a cellular vaccine, are currently considered to be useful for treating
cancer. An important step in the production of these hybridomas is quantification
of the yield. In the past, the yield of hybridomas was determined by labeling the
two cell types with cytoplasmic fluorescent dyes. However, it would be ideal if
the hybridoma yield could be obtained using a strategy with a more biologically
relevant process, such as antigen presentation. In this study we monitored the
average percentage of hybridoma cells by measuring the extent of lysosomal fusion.
Lysosomes are dynamic organelles that undergo self- or homotypic fusion, which is
involved in antigen presentation. By using confocal microscopy, the level of fused,
fluorescently labeled (red and green) lysosomes in a hybridoma cell was determined
by measuring the colocalization of red and green pixels (appearing yellow) relative
to all red and green pixels in the image. The results show that lysosomal fusion
occurs in electrofused cells and that this is an extensive, time- and temperaturedependent
process. This approach provides a new method for optimization of the
preparation of hybridoma cell vaccines, based on the intracellular process of antigen
presentation.
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