Eclipse ti e a1r si

Oxidative stress analysis and cell morphology were conducted by using the laser confocal microscope Nikon Eclipse Ti-E A1R-Si controlled by Nikon NIS Elements AR software (Nikon Instruments, Amsterdam, The Netherlands). Fibroblasts were cultured in 4-well chamber slides. After exposure to LF and UVA radiation, according to the procedure presented in Section 2.2, cells were incubated with CellROX^® Green Reagent (5 μM, Thermo Fisher Scientific, Waltham, MA, USA) and Hoechst 33342 (5 μg/mL, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min., washed thrice in PBS, and imaged. Hoechst 33342 stains nuclei and CellROX^® dye were used for detecting oxidative stress. CellROX^® Green Reagent in the oxidized form is a bright green fluorescent, while in the reduced state it exhibits very weak fluorescence. The confocal imaging in transmitted light was applied to estimate the morphology of the cells.

Imaging of melanoma cells was performed using the laser confocal microscope Nikon Eclipse Ti-E A1R-Si and Nikon NIS Elements AR software. A375 and COLO 829 cells were cultured in sterile cover slips in Petri dishes. After 48-h exposure to tigecycline, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Afterwards, fixed cells were treated with glycine and BSA solutions and then were incubated with primary anti-p16 (1:800) and anti-p21 (1:400) rabbit antibodies overnight at 4 °C. In the next step, the samples were stained with Alexa Fluor 488 conjugated with the secondary antibody (1:200), SYTO Deep Red Nucleic Acid Stain (1:100), and Phalloidin–Atto 565 (0.6 µM). The reagents allowed for imaging of p16 and p21 proteins, as well as nucleus and actin cytoskeleton, respectively. Finally, fixed and stained cells on cover slips were mounted onto microscopic glass slides.

Imaging of MCF-7 and MDA-MB-231 cells was performed using the laser confocal microscope Nikon Eclipse Ti-E A1R-Si and Nikon NIS Elements AR software. The cells were cultured in sterile cover slips placed in Petri dishes to 80% confluence. Next, the cells were treated with the tested compounds. The examined cells were fixed with paraformaldehyde (4%). Cell membranes were permeabilized with 0.1% Triton X-100. All samples were blocked with glycine and BSA solutions and then incubated overnight at 4 °C with rabbit antibody conjugated with Phycoerythrin and Alexa Fluor 488: anti-p53 (1:50). Afterward, cells were stained with DAPI (1:500) and Phalloidin–Atto 565 (0.6 µM). The final staining allowed imaging the nucleus and actin filaments, respectively. Finally, the cover slips were mounted onto microscopic glass slides.

The assessment of the influence of minocycline on human melanoma cell abundance and morphology was carried out after 24 h-long treatment. The cells were cultured in sterile cover slips placed in Petri dishes. After the exposure to minocycline in concentrations of 50 µM, 100 µM, and 200 µM, the cells were fixed with paraformaldehyde (4%), washed three times with PBS, and permeabilized with 0.1% Triton X-100 solution. In the next step, cells were treated with 0.25% glycine solution for 30 min and then incubated overnight with the primary tyrosinase monoclonal antibody. Afterward, the tested cells were washed three times with PBS and stained with 1 µM of SYTO (specific for DNA), 0.6 µM of Phalloidin–Atto 565 (specific for actin filaments), and 10 µg/mL of the secondary antibody conjugated with Alexa Fluor 488 (tyrosinase).
After the staining procedure, the cover slips were mounted onto microscopic glass slides. The samples were imaged by means of the laser confocal microscope Nikon Eclipse Ti-E A1R-Si and Nikon NIS Elements AR software.

Laser confocal microscope Nikon Eclipse Ti-E A1R-Si and Nikon NIS Elements AR software were used to cell visualization. Melanoma cells were cultured on sterile coverslips in Petri dishes in the recommended growth media. After the treatment procedure, the cells were fixed and permeabilized with 4% paraformaldehyde and 0.1% Triton X-100, respectively. Before the staining, samples were incubated with glycine and bovine serum albumin. Afterwards, the cells were incubated with primary rabbit anti-p53 antibody (1:1000) (Cell Signaling, Danvers, MA, USA) at 4 °C for 24 h. Then, the samples were stained with Alexa Fluor 488-conjugated anti-rabbit secondary antibody (1:200) (Thermo Fisher Scientific, Waltham, MA, USA). The labeling of cell nuclei with SYTO Deep Red Nucleic Acid Stain (Thermo Fisher Scientific, Waltham, MA, USA) as well as labeling of actin filaments with Phalloidin–Atto 565 (Sigma-Aldrich Inc., Taufkirchen, Germany) were also applied according to the manufacturer’s protocol.

Imaging of darkly pigmented human melanocytes were performed using light inverted microscope NIKON TS100F (Japan) as well as the laser confocal microscope Nikon Eclipse Ti-E A1R-Si and Nikon NIS Elements AR software. The cells were cultured in sterile cover slips placed in Petri dishes for 48 h. Next, the cells were treated with minocycline and exposed to UVA and UVB radiation, according to paragraph 5.3. The exanimated cells were fixed with paraformaldehyde (4%) and permeabilized with 0.1% Triton X-100 6 h after the irradiation procedure. All samples were blocked with glycine and BSA solutions and then were incubated with primary anti-tyrosinase antibody (1:100) overnight at 4 °C. Afterward, melanocytes were stained with DAPI (1:500), Phalloidin–Atto 565 (0.6 µM) and conjugated with the secondary antibody-Alexa Fluor 488 (1:200). The staining allowed to image nucleus, actin filaments and tyrosinase, respectively. Finally, the cover slips were mounted onto microscopic glass slides.