Calcein am pi double stain kit

A Calcein-AM/PI Double Stain Kit (Solarbio, Beijing, China) was used according to the manufacturer’s instructions. Calcein-AM (2 μmol/l) and propidium iodide (4.5 μmol/l) were added to the culture wells and incubated at 37 °C in the dark for 15 min. Living cells (green cytoplasmic fluorescence) and dead cells (red nucleus) were observed with an inverted fluorescence microscope (Nikon, Tokyo, Japan).

Dichloromethane (99.5%) was purchased from Haohua Chemical Reagent Co. Ltd. (Luoyang, China). Lsopropyl alcohol (99.7%) was obtained from Tianli Chemical Reagent Co. Ltd. (Tianjin, China). DNase/RNase-Free Water (DEPC water) and Calcein-AM/PI double stain kit were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). Annexin V/PI kit was purchased from Bio-Box. Cell Counting Kit-8 (CCK-8), Reactive Oxygen Species Assay Kit (ROS Assay Kit) and Mitochondrial membrane potential assay kit (JC-1) were bought from Beyotime Biotechnology. Cell culture media (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), and antibiotics were obtained from Biological Industries. All chemicals were of analytical or equivalent grade and used without further treatment.

Based on ISO10993-12 standard, the extraction DMEM medium was obtained at 3 cm²/mL (volume ratio of NiTi surface area to extraction liquid). To evaluate cell proliferation activity, mouse fibroblast L-929 cells were seeded in 96-well plates at a density of 5×10³ cells/well and cultured in an incubator at 37 °C for 24 h. The cell lines present in this study were obtained from Zhongqiao Xinzhou Co., Ltd., Shanghai China. 100 μL extract from different samples was added to each well, and DMEM medium was added to the blank control group. After 1, 3, and 5 days of co-culture, the reagents were added according to the instructions of CCK-8 (Dojindo, Japan) and incubated for 1 h. The absorbance at 450 nm was measured. Live/dead staining assays were used to assess the morphology of L-929 cells. Cells were cultured with extract for 5 days and stained with a Calcein-AM/PI Double Stain Kit (Solarbio, China). Then, cells were imaged under an inverted fluorescence microscope (DMI8A, Leica). To evaluate ROS production, L-929 cells were inoculated into 6-well plates at a density of 2×10⁵ cells per well, and extracts of different groups were added. After 24 h of co-culture, a diluted 100 μL DCFH-DA (Sigma-Aldrich, United States) probe was added to each well. After incubation, the fluorescence intensity was detected by flow cytometry (ThermoFisher DxFLEX, United States).

PTX, dithiothreitol (DTT), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR), and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) were supplied from Meilun Biotech (Dalian, China). Perfluorooctanol, octanol, coumarin-6, 1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDCI), 4-dimethylaminopyrideine (DMAP) and hydrogen peroxide (H₂O₂) were all bought from Aladdin Biochemical Technology Co. Ltd. (Shanghai, China). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methyl (polyethylene glycol)-2000 (DSPE-PEG_2k) was provided from Shanghai Advanced Vehicle Technology Co. Ltd. (Shanghai, China). Cell culture reagents and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were derived from Gibco (Beijing, China). Calcein-AM/PI double stain kit, Annexin V-FITC/PI apoptosis assay kit and Microtubule Tracker Red were purchased from Beijing Solarbio Science & Technology Co., Ltd. TUNEL apoptosis detection kit and Ki67 cell proliferation kit were supplied from Service biotech Co., Ltd. (China). Hoechst 33342 was provided by BD Biosciences (USA). The other reagents used in this paper were of analytical.

Cells were seeded on 35-mm confocal dishes precoated with scaffolds at a density of ~1 × 10⁵ cells per well. Then, the cells were stained with a calcein AM/PI double-stain kit (Solarbio, Cat#1630) according to the instructions of the manufacturer for CLSM imaging. All the samples were observed by a Leica SP8 laser scanning microscope.

1, 3-diphenylisobenzofuran (DPBF) was purchased from Alfa Aesar (Tianjin, China). 2,2,6,6-tetramethylpiperidine (TEMP) was purchased from Meryer (Shanghai, China). Cell Counting Kit-8 was purchased from Changchun Sanbang Pharmaceutical Technology Co., Ltd. (Changchun, China). DCFH-DA and Calcein-AM/PI double stain kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). IR820 was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). All of the chemicals and solvents were used as received without further purification.

The effects of paeonol on the viability or cytotoxicity of macrophages cells were assayed using the Calcein-AM/PI Double Stain Kit (Solarbio, Beijing, China). In brief, RAW264.7 cells were exposed to P. aeruginosa in DMEM without antibiotics and treated with paeonol. The cells washed with PBS three times and stained with a 100 μL Calcein-AM/PI stain solution at 37°C for 15 min. Living cells with green cytoplasmic fluorescence and dead cells with red nuclei were immediately observed by the fluorescence microscope.