Lsm 880 spectral confocal laser scanning microscope

Raw 264.7 cells were incubated with LPS for 1 and 24 h (200 ng/ml) in order to induce activation. ER tracker (catalog no.: E34250; Invitrogen) was used at 1 μM final concentration in the cell medium and was incubated 30 min before fixation. Peritoneal cells were centrifuged in cytospin after peritoneal lavage. In all cases, cells were fixed with 4% paraformaldehyde for 15 min at room temperature (RT), permeabilized, and blocked (1% bovine serum albumin, 0.1% saponin, 150 mM glycine, and 5% FBS in PBS) for 1 h at RT. Primary antibodies against SIRT6 and TNFα were prepared in antibody dilution buffer (1% bovine serum albumin, 0.1% saponin, and 150 mM glycine in PBS) and incubated overnight at 4 °C. Alexa-conjugated secondary antibodies were incubated for 1 h. Nuclei were stained using 4′,6-diamidino-2-phenylindole (catalog no.: D9542; Sigma) and actin filaments with phalloidin (catalog no.: A22283; Invitrogen). Samples were mounted in Prolong Gold Antifade Reagent (catalog no.: P10144; Invitrogen), and images were acquired using a Zeiss LSM 880 spectral confocal laser scanning microscope, using a 60× oil-immersion objective (numerical aperture of 1.45). Images were processed using ImageJ (Wayne Rasband, National Institutes of Health).

A549 cells were cultured (3 × 10⁴ cells/well) in an 8-well sterile µ-Slide (Ibidi, Gräfelfing, Germany). The next day, the cells were treated with compound 1 (at both 2 and 20 µM) or with compound 2 (at 20 µM) for 1 h. Afterwards, the cells were irradiated (pre-irradiated) or not (dark conditions) with 700–400 nm light for 1 additional hour. In some experiments, the cells under dark conditions were irradiated for 5 min with the 488 nm fluorescent laser from a confocal microscope. To localize the drug inside the cells, it was excited with 405 nm laser and the emission between 594 and 754 nm was recorded. The differential interference contrast (DIC) image was used to characterize the different cellular structures. Images were taken using a Carl Zeiss LSM 880 spectral confocal laser scanning microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) and analyzed with ZEN 2 blue edition software (Zeiss). Representative images from three independent experiments are shown.

HEK-293T cells were grown on glass coverslips and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were blocked and permeabilized with 0.5% bovine serum albumin and 0.05% Saponin in PBS for 20 minutes. The primary antibodies, anti-myc (1:100), anti-C-RAF (1:100) and anti-HERC1 (410; 1:100), were incubated at 37°C for 1 hour. After washing, secondary antibodies (1:500) were incubated at 37°C for 45 minutes. F-actin was detected incubating for 20 minutes at room temperature with Phalloidin-Alexa 647 (100 ng/ml). Nuclei were stained with DAPI (1 μg/ml). Images from optical sections (thickness: 0,31 μm) were acquired using a Carl Zeiss LSM 880 spectral confocal laser scanning microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) using a 63x oil immersion objective (1.4 numerical aperture) and image resolution of 1024 X 1024 pixels. Pearson’s correlation coefficient (POC) and Manders’ overlap coefficient (MOC) were calculated using ImageJ software.