2.11.3. Fluorescence Microscopy Based Cell Uptake Study

After completion of cytotoxic assay, the fluorescence-based cell uptake was detected through fluorescence microscopy at image station of University of Lahore by Confocal Laser Scanning microscope (Leica TCS SP5 Inverted Supercontinuum X, Leica Microsystems, Chicago, IL, USA). Initially the HepG2 cells were seeded in multi-chambered (eight chambered) glass bottom dishes containing 10,000 cells per dish at 37 °C for 24 h and then the growth media was removed and the cells were washed with Hank’s balanced salt solution-HEPES (HBSS-HEPES, pH 7.4) buffer. After that Dox solution and Dox loaded liposomes using 10 µg Dox/mL concentration were incorporated in each dish and incubated at 37 °C for preselected time spans. The RPMI media was considered as negative control. The cells were washed for multiple times to remove free nanoparticles and then the nuclei staining of HepG2 cells was done by incubating the cells with DAPI blue for 5 min at 25 °C. The extra amount of stain was removed by multiple washings with PBS and then the cells were fixed by using the formaldehyde solution. The samples were visualized under confocal laser scanning microscope.

The aforementioned procedure was repeated for the above-mentioned samples at mild hyperthermia (40.2 °C) through empty incubator for 1 h as well (as discussed earlier). The aforementioned samples were then observed through fluorescence microscope.