Gfp 3035d

All live-cell microscopy experiments were performed on an Olympus IX73 inverted microscope if not mentioned otherwise. The microscope is equipped with an UApoN340 40× oil immersion objective (Olympus, Tokyo, Japan) and a CCD Retiga R1 camera (Q-imaging, Tucson, AZ, Canada). For illumination, a LedHUB^® (Omicron, Vienna, Germany) equipped with 340, 385, 455, 470, and 550 nm LEDs in combination with CFP/YFP/RFP (CFP/YFP/mCherry-3X, Semrock, Rochester, NY, USA) or GFP (GFP-3035D, Semrock, Rochester, NY, USA) filter set was used. Alternatively, an AnglerFish F-G/O (NGFI, Grraz, Austria) has been used. Data acquisition and control of the fluorescence microscope were performed using Visiview 4.2.01 (Visitron, Puchheim, Germany).

All live-cell microscopy experiments were performed on an Olympus IX73 inverted microscope if not mentioned otherwise. The microscope is equipped with an UApoN340 40× oil immersion objective (Olympus, Japan) and a CCD Retiga R1 camera (Q-imaging, Canada). For illumination, a LedHUB® (Omnicron, Germany) equipped with 340, 385, 455, 470, and 550 nm LEDs in combination with CFP/YFP/RFP (CFP/YFP/mCherry-3X, Semrock, USA) or GFP (GFP-3035D, Semrock, USA) filter set was used. During the measurements cells were continuously perfused by a gravity-based perfusion system (NGFI, Graz, Austria). Data acquisition and control of the fluorescence microscope was performed using Visiview 4.2.01 (Visitron, Germany).

Apoptosis level was analyzed using an Apoptosis/Necrosis detection kit (ab176749, Abcam, Cambridge, UK). The cells were seeded at a density of 15,000 per well of a 96-well plate and grown for 12 h. After that, 475 µM of H₂O₂ alone or with the peptide was added in 100 µL of the fresh medium to 100 µL of the old medium in the wells and incubated for 1 h at 37 °C in a CO₂ incubator. After that, the medium was removed, and the cells were stained according to the manufacturer’s instructions using the phosphatidylserine sensor (apoptotic cells, green fluorescence) and membrane-impermeable dye 7-AAD (necrotic cells, red fluorescence). The stained cells were photographed using an inverted fluorescent microscope Nikon Ti-S using a Semrock GFP-3035D filter cube with magnification 100×. For each well, five non-intersecting view fields were captured, and apoptotic cells were counted.