A novel assay for analysis of adenovirusmediated endosome lysis of T lymphocytes

Abstract:

[Objective] To analyze adenovirus-mediated endosome lysis of T cells, we developed a novel approach based on pHrodo dextran (pH-sensitive fluorescent dye).[Methods] After incubating Jurkat cells (T cell leukemia) with serotype 5 adenovirus (Ad5) and pHrodo dextran, we determined the optimal incubation time and concentration of pHrodo dextran. To assess viral lysis of the endosome, we monitored the ratio changes of mean fluorescence intensity in different time points by laser scanning confocal microscopy.[Results] After incubating Jurkat cells with Ad5 and 80 μg/mL pHrodo dextran for 10 minutes, we observed the fluorescence intensity was significantly reduced at 30 minutes compared with that of endosomes at 0 minute. However, we found the mean fluorescence intensity was only slightly reduced by inhibiting V-ATPase with the bafilomycin A1 treatment.[Conclusion] The method based on pH-sensitive dye can be used to analyze the adenovirus-mediated endosome lysis of T cells.

Key words:adenovirus;endsome;intracellular trafficking;T lymphocytes