Hc pl apo 63 1.4 objective

HeLa cells were seeded in 8-well Ibidi μ-slides (Ibidi GmbH, Germany) at a density of 20,000 cells/well 24 h prior to the experiment. On the next day, the medium was replaced with 270 μL of fresh medium. A total of 30 μL of carboxyfluorescein-labeled IGPNs was added to each well, corresponding to a final concentration of 10 μM peptides. The medium was removed after 4 h incubation, and the cells were washed twice with 300 μL of PBS followed by 40 min of fixation with 4% PFA at RT. The cells were then washed twice with PBS again, and the cell nuclei were stained with 2 ng/μL DAPI. The DAPI solution was discarded after 20 min incubation, and the cells were further washed with trypan blue (0.04%, w/v). Next, 300 μL of PBS was added per well for CLSM imaging. Images were recorded on a Leica-TCS-SP8 confocal laser scanning microscope equipped with a HC PL APO 63 × 1.4 objective (Germany). DAPI and carboxyfluorescein emission were recorded at 450 nm and 520 nm, respectively. All images were analyzed using the LAS X software from Leica.

HeLa cells were seeded in 8-well Ibidi μ-slides (Ibidi GmbH, Gräfelfing, Germany) at a density of 20,000 cells/well 24 h prior to the treatment. On the next day, the medium was replaced with 270 μL of fresh medium. A total of 30 μL of IGPNs and peptides was added to each well, resulting in a final concentration of 10 μM peptides, and the cells were incubated for 24 h. As a positive control, tert-butyl hydroperoxide solution (TBHP, 200 μM) was added, and the cells were incubated for 30 min. The cells were then washed twice with PBS followed by 30 min of staining with H₂DCFDA (10 μM) in the dark. Next, the H₂DCFDA solution was discarded, and 300 μL of PBS was added per well for CLSM imaging. Images were recorded on a Leica-TCS-SP8 confocal laser scanning microscope equipped with a HC PL APO 63× 1.4 objective (Germany). The H₂DCFDA signal was recorded with emission at 520 nm. All images were analyzed using the LAS X software from Leica.