4.8. Lipid Droplets Staining

Lipid droplet formation in response to PF-06424439 pre-treatment and X-ray irradiation was detected using the lipophilic dye LD540 [14]. Cells were cultivated in 6-well plates at a density of 1.4 × 10⁵ cells/ well on coverslips, treated with 10 μM of PF-06424439 for 72 h and then irradiated with 0 and 6 Gy. After 30 min and 72 h from irradiation, cells were fixed with 4% PFA for 45 min at room temperature. Cells were rinsed twice with cold DPBS and then stained with LD540 diluted 1:5000 in DPBS, in the dark. The cells were then washed two times and counterstained with Hoechst 33342 at a final concentration of 1 μg/mL for 20 min. Two further washes with DPBS were carried out and then coverslips were mounted on glass slides using SlowFade Diamond Antifade Mountant. Zeiss LSM710 confocal-laser-scanning microscope with 63× oil immersion objective was used to detect LDs in all the samples. ImageJ 1.52p software was used for processing the confocal images in Z-stacks and counting LDs.