Calcein am pi live cell dead cell double staining kit

After the primary hippocampal neurons were cultured to the seventh day, the Calcein-AM/PI live cell/dead cell double staining kit (CA1630, Solarbio, Beijing, China) was used to evaluate the inhibitory effect of pioglitazone on neuronal ferroptosis, and the determination was carried out according to the manufacturer’s instructions (Karuppagounder et al., 2018 (link)).

For the apoptosis experiment of PC12 cells, PC12 cells were loaded in each upper chamber and stimulated with 200 μM hydrogen peroxide (H₂O₂) for 12 h to simulate damage to cells caused by oxidative stress. After changing to standard medium, primary M1 macrophages were placed in the lower layer of the Transwell™ coculture model for 12 h. Different nanoparticles (PBS, EVs, NGF, EVs^−NGF, EVs^−cl−NGF) were added to the different chambers, and after 12 h, the apoptosis rate of PC12 cells and the macrophage polarization level in each chamber were evaluated by flow cytometry and CLSM.
For the Live-Dead cell staining experiment, PC12 cells were collected by digestion centrifugation and prepared into a cell suspension. 100 µl staining working solution (Prepared according to Calcein-AM/PI Live Cell/Dead Cell Double Staining Kit (Solarbio, Beijing, China) instructions) was added to 200 µl cell suspension and incubated at 37 °C for 15 min. Live cells (green fluorescence, 488 nm) and dead cells (red fluorescence, 550 nm) are simultaneously detected under CLSM.

Iron acetylacetonate (Fe(C₅H₇O₂)₃), zinc nitrate (Zn(NO₃)₂), p-phthalic acid (C₈H₆O₄), polyvinyl pyrrolidone (PVP), ethanol (C₂H₆O) and N, N-Dimethylformamide (DMF) were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. 9,10-Anthracenediyl-bis (methylene) di malonic Acid (ABDA), Indocyanine green (ICG) was purchased from Shanghai Macklin Biochemical Co., Ltd. Phosphate-buffered saline (PBS), Dulbecco’s modified eagle medium (DMEM), Fetal bovine serum (FBS), sodium pyruvate, essential amino acids, and non-essential amino acids were from ExCell Bio (China). Calcein-AM/PI Live Cell/Dead Cell Double Staining Kit was purchased from Beijing solarbio science & technology co.,ltd. All other reagents were from Beyotime Biotechnology (China) and used as received unless otherwise noted.