Phosphate buffered saline pbs 1x

The evaluation of nuclear localization and nuclear changes indicative of apoptosis (i.e. nuclear shrinkage/fragmentation) was performed using the 4,6′-diamidino-2-phenylindole (DAPI) Staining. HaCaT and A375 cells were seeded in 6-well plates at an initial cell density of 1 × 10⁶ cells/well. After cells reached 85–90% confluence, the old medium was removed and replaced with a fresh medium containing the highest concentrations of OA, UA, and BA derivatives (1, 2, and 3 50 μM) and BA, OA, and UA, respectively. The cells were treated with the tested compounds and incubated for 24 h at 37 °C. After the incubation period, the staining protocol was performed through the following steps: the cells were washed 2–3 times with cold phosphate-buffered saline PBS (1X) (Thermo Fisher Scientific, Boston, MA, USA), fixed up with paraformaldehyde 4% in PBS and then permeabilized with Triton X/PBS 2% for 30 min at room temperature. After another washing step with cold PBS, the cells were blocked with 30% FCS in 0.01% Triton X for 1 h at room temperature. In the end, the cells were washed again 2–3 times with cold PBS, stained with a solution of 4,6′-diamidino-2-phenylindole (DAPI, 300 nM), and incubated at 4 °C in the dark overnight. The nuclear alterations were analyzed using the integrated DP74 digital camera of an Olympus IX73 inverted microscope (Olympus, Tokyo, Japan).

The morphological assessment of apoptotic cells in terms of nuclear localization and nuclear changes indicative of apoptosis (i.e., nuclear shrinkage/fragmentation) was performed using the 4,6′-Diamidino-2-Phenylindole (DAPI) staining. HaCaT and A375 cells were seeded in 6-well plates (1 × 10⁶ cells/well) and incubated until reaching 85–90% confluence. The old medium was then replaced with fresh medium, and the cells were stimulated with GNP and triterpene GNP conjugates (10, 25 and 50 μΜ) and incubated for another 24 h at 37 °C. After the stimulation period, the cells were washed 2–3 times with cold phosphate buffered saline PBS (1X) (Thermo Fisher Scientific, Boston, MA, USA) and fixed with paraformaldehyde 4% in PBS. The cells were then permeabilized with Triton X/PBS 2% for 30 min at room temperature, washed 2–3 times with cold PBS and blocked with 30% FCS in 0.01% Triton X for 1 h at room temperature. After another washing step with cold PBS, the cells were stained with DAPI (300 nM) and incubated at 4 °C in the dark overnight. The nuclear alterations were analyzed using a Zeiss Microscope AXIO Observer.D1 (Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with the Snap-260—ZEN pro 2012.