Monitoring lysosomal fusion in electrofused hybridoma cells.
Dendritic and tumor cells are fused to produce hybridoma cells, which are considered to be used as cellular vaccines to treat cancer. Previous strategies for hybridoma cell production were based on the quantification of the electrofusion yield by labeling the cytoplasm of both parental cell types. However, a better physiological strategy would be to label subcellular structures related directly to the antigen presentation process. Therefore, we here electrofused the same amount of CHO cells stained with red and green fluorescent dextrans and have monitored the yield of hybridoma cell formation by measuring the fusion of red and green late endocytic organelles that are involved in antigen presentation. By using confocal microscopy, the level of fused, fluorescently labelled late endocytic compartments in a single hybridoma cell was determined. The results demonstrate that organellar fusion occurs in hybridomas, which is time- and temperature-dependent. This approach therefore provides a new method for the hybridoma cell vaccine evaluation, which is based on the intracellular physiological mechanism of antigen presentation.
Gabrijel
Mateja
M
Celica Biomedical Center, Proletarska cesta 4, 1000 Ljubljana, Slovenia.
Kreft
Marko
M
Zorec
Robert
R
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004560:Electricity; D004705:Endocytosis; D006825:Hybridomas; D007425:Intracellular Membranes; D008247:Lysosomes; D008561:Membrane Fusion; D018613:Microscopy, Confocal; D013696:Temperature