Bafilomycin a1 from streptomyces griseus

Cells were pre-treated with inhibitors and, subsequently, with DMPC/1a or DMPC/1b liposomes loaded with m-THPC, for 1 h. The inhibition of clathrin function was achieved by pretreating LN229 cells with 28 μM chlorpromazine for 60 min at 37 °C. To investigate the caveolae-mediated uptake, cells were treated with 9 μg mL -1 of Filipin III from Streptomyces filipinensis for 60 min at 37 °C. In order to investigate the dependence of liposome uptake on the acidification of endosomes, cells were treated with 100 nM Bafilomycin A1 from Streptomyces griseus (Sigma-Aldrich) for 60 min at 37 °C. The role of the actin cytoskeleton in endocytosis was investigated by pre-treating cells with 30 μM LY294002 for 60 min at 37 °C.
At the end of each treatment, cells were washed with icecold Hank's balanced salt solution (HBSS), detached with EDTA and 0.25% trypsin, resuspended in ice-cold PBS and immediately analyzed for the photosensitizer content. filter and acquired in "log" mode. At least 10 000 events were analyzed. The m-THPC content was evaluated as fluorescence intensity, expressed as the mean fluorescence channel (MFC). The analysis was performed using CellQuest™ software (Becton Dickinson). Results analyzed were reported as mean percent of inhibition obtained from 3 independent experiments.