Lsm 710 axio obzerver z1 duo nlo laser scanning microscope

Intracellular distribution of PS and PD was studied by using the LSM 710 Axio Obzerver Z1 DUO NLO laser scanning microscope (Carl Zeiss, Germany). The images were obtained using a LD C-Apochromat water immersion objective lens 40×/1.1. The GL261 cells were seeded in 96-well glass-bottom plates (Corning, USA) at 10⁴ cells per well and grown overnight. The cells were then incubated with 10 μM photosensitizers in serum-free culture medium for 1–4 h, followed by washing with PBS and confocal image acquisition. The fluorescence of PS and PD was excited at 633 nm and recorded in the range of 650–735 nm.
For colocalization analysis of PS and PD after 3.5 h of incubation of GL261 cells with the respective photosensitizer, the following dyes were added for 30 min (ThermoFisherScientific): 0.5 μM LysoTracker Green DND-26 for lysosomes, 0.5 μM ER-Tracker for endoplasmic reiculum, 0.5 μM MitoTracker Green FM for mitochondria, 5 μM BODIPY FL C5-ceramide complexed to BSA for Golgi apparatus. Dyes were added to living cells that had been incubated with the photosensitizers. Staining was performed according to the manufacturer’s instructions. Fluorescence of stained organelles was excited by an argon laser at 488 nm and registered in the range of 500–560 nm.

Glioma GL261 cells were seeded in 96-well glass-bottom plates (Corning Inc., Corning, NY, USA) at 1 × 10⁴ cells per well and grown overnight (24 h). Next, the glioma cells were incubated in a serum-free medium supplemented with 10 μM pz I or pz III for 4 h, followed by washing with phosphate-buffered saline (PBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA). To analyze the intracellular distribution of pz I and pz III, the following dyes were added 30 min before the end of the incubation period: LysoTracker Green DND-26 for lysosomes (0.5 μM), ERTracker for endoplasmic reticulum (ER, 0.5 μM), MitoTracker Green FM for mitochondria (0.5 μM), BODIPY FL C5-ceramide complexed with bovine serum albumin for Golgi apparatus (5 μM), and DAPI for nucleus (2.8 μM) (all fluorescent dyes were purchased from Thermo Fisher Scientific, Waltham, MA, USA). The stained glioma GL261 cultures were observed in an LSM 710 Axio Obzerver Z1 DUO NLO laser scanning microscope (Carl Zeiss, Oberkochen, Germany) with an LD C-Apochromat water immersion objective lens, 40×/1.1. Fluorescence of the stained organelles was excited using an argon laser at 488 nm and recorded at 500–560 nm [26 (link)].