Confocal fluorescence imaging

0.3 × 10⁶ A549 cells were seeded onto sterile glass coverslips in a 6-well plate in supplemented RPMI. 24 hours post seeding, RPMI was removed from the cells and cells were washed with DPBS. Cells were then incubated at 37 °C and 5% CO₂ in one of the following: serum-free DMEM for 1 hour; 100 ng/mL Verteporfin in serum-free DMEM for 1 hour; 10 mM 5-ALA in serum-free DMEM for 3 hours; 10 ng/mL Temoporfin in serum-free DMEM for 24 hours. Following incubation, photosensitiser solutions were removed and cells washed with DPBS. Cells were then fixed with 4% (v/v) PFA solution for 20 minutes at room temperature. After fixation, PFA solution was removed and cells washed twice with DPBS. Cell staining (under dark conditions) was performed as follows. DPBS was removed from cells and cells were incubated with 0.2% Triton-X in DPBS for 10 minutes at room temperature. Triton-X was removed from cells and cells washed three times with DPBS. Cells were then incubated with 5% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich) for 1 hour at room temperature. BSA was removed from cells and cells washed twice with DPBS. Cells were then incubated with AlexaFluor 488-conjugated Phalloidin (Thermo Fisher), 1:40 in DPBS for 45 minutes at room temperature. Solution was removed and cells were washed 5 times for 5 minutes each time with 0.2% (v/v) Tween 20 (Sigma-Aldrich) in DPBS. Cells were then washed twice with DPBS. Coverslips were then removed from 6-well plate and mounted onto microscope slides (VWR) using Fluoromount (Serva) and allowed to dry before imaging. Imaging was performed using a Leica SP5 inverted confocal microscope equipped with a 405 nm diode laser and a 20x objective. Excitation was performed at 405 nm, with collection from 630 nm to 800 nm.