Annexin 5 pi staining protocol

Apoptosis was evaluated using the Alexa Fluor^® 488 Annexin V/Dead Cell Apoptosis Kit with Alexa Fluor 488 Annexin V and propidium iodide (PI) for flow cytometry (Life Technologies, Carlsbad, CA, USA). The MCF-7, Colo205, and HepG2 cells (1 × 10⁵ cells per well) were incubated for 24 h. Subsequently, the medium was removed, and GO (100 µg/mL), NP-Pt (25 µg/mL), and GO-NP-Pt (GO₁₀₀:Pt₂₅ µg/mL) in the culture medium were added to the cells and incubated for an additional 24 h. The positive control was prepared as described in Jaworski et al. [26 (link)] After this, the MCF-7, Colo 205, and HepG2 cells were harvested, washed in cold PBS, transferred to tubes, and stained according to the Annexin V/PI staining protocol (Life Technologies). Cancer cells were analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA), measuring the fluorescence emission at 530 nm and 575 nm (or equivalent), using excitation at 488 nm. Positive cells were identified on the basis of the fluorescence intensity of Annexin V-Alexa Fluor 488 (early stage of apoptosis) or PI (end stage of apoptosis and necrosis). Data were analyzed using the Cell Quest Pro software ver. 5.1 (Becton Dickinson), and the regions were set on the basis of positive and negative control samples.