Macs quantify flow cytometer

Intracellular ROS levels were determined using a ROS detection assay kit (Beyotime Biotechnology, Shanghai, China). T24 and 5637 cells were treated with CONPs at a concentration of 0.625, 1.25, or 2.5 mg/ml for 24 h. In inhibitory studies, the cells were incubated with CONPs alongside 5 mM of the ROS inhibitor N-acetylcysteine (NAC) (Beyotime Biotechnology) or 10 mM of 3-MA autophagy inhibitor (Sigma-Aldrich) for 2 h. The cells were then harvested, washed three times with PBS, and treated with 10 mM dihydrodichlorofluorescein diacetate (DCF-DA) in serum-free McCoy’s 5 A medium at 37 °C for 30 min. Then, the cells were washed 3 times with serum-free medium, after which DCF fluorescence was measured by using a MACS Quantify^TM flow cytometer (Miltenyi Biotec, Germany).

Apoptosis and necrosis were analyzed with the Annexin V–fluorescein isothiocyanate apoptosis detection kit (Lianke Biotech) following the manufacturer’s instructions. Cell cycle assay was assessed by flow cytometry using a cell cycle staining kit (Lianke Biotech) according to the manufacturer’s standard protocol. The samples were analyzed using a MACS Quantify^TM flow cytometer (Miltenyi Biotec), and the results were analyzed using the FlowJo v7.6.1 software (https://www.flowjo.com/).