Annexin 5 pe 7 aad apoptosis detection kit

CD11b and CD14, markers for cell differentiation, were detected by flow cytometry. Cells were seeded at a density of 1×10⁶ cells per well in 6-well culture plates and treated with different concentrations (0 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM) of ATO. On days 0, 3, 6, 9 and 12 of treatment, the cells were collected and incubated with CD11b or CD14 for 30 minutes, and examined by BD FACS LSR II flow cytometer. The IgG antibody treated group was used as a negative control. The percentage of CD11b-positive cells or CD14-positive cells was analyzed using FlowJo software, version 7.6.1.
Apoptosis was detected by flow cytometry using Annexin V-PE/7-AAD Apoptosis Detection Kit (MultiSciences Biotech Co., Ltd, Hangzhou, China). Cells were seeded 4×10⁵ cells per well in 6-well culture plates and incubated with different concentrations (0 μM, 2 μM, 4 μM, 8 μM) of ATO for 24h or 48h. Then, cells were collected and incubated with AnnexinV-PE and 7-AAD according to the manufacturer's instructions. The cells were examined and analyzed as described above.

Apoptosis was detected by flow cytometric analysis using an Annexin V-PE/7-AAD Apoptosis Detection Kit (MultiSciences Biotech Co., Ltd, Hangzhou, China) according to the protocol provided. Briefly, the cells were seeded (3×10⁵ cells/well) in a 6-well plate and incubated for 48 h. All groups were treated with staurosporine (Sigma, St. Louis, MO, USA) (2 µM for Bcap-37 cells, 100 nM for MDA-MB-231, 1 µM for MCF-7 and SK-BR-3), an inducer of apoptotis, as a positive control. Then, the treated cells were harvested and incubated with Annexin V-PE and 7-AAD for 15 min at room temperature in the dark and immediately analyzed by flow cytometry (FACScalibur flow cytometer, BD, CA, USA). Each experiment was carried out for at least three times.

Horseradish peroxidase (HRP)-conjugated anti-rabbit, anti-mouse immunoglobulin G, penicillin–streptomycin solution, Bradford protein assay kit, Cell counting kit-8 (CCK-8 kit), mitochondria membrane potential assay kit (JC-1), and LDH Release Assay Kit were obtained from Beyotime (Shanghai, China). Annexin V–PE/7AAD apoptosis detection kit was purchased from MULTI SCIENCES. Giemsa and crystal violet were purchased from Solarbio Bioscience and Technology (Shanghai, China). Trypan blue was obtained from Life Technologies (Carlsbad, CA, United States). BCA protein assay kit and Pierce ECL western blotting substrate were obtained from Thermo Fisher Scientific (MA, United States). Intact cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) assay kits were purchased from Seahorse Bioscience Company (North Billerica, MA, United States). Glucose was obtained from Sigma (St. Louis, MO, United States). L-glutamine was bought from Sangon Biotech (Shanghai, China). Puromycin was obtained from Amresco (WA, United States). Protease (Complete Mini) and phosphatase (PhosphoSTOP) inhibitor cocktail tablets were purchased from Roche Applied Science (Indianapolis, IN, United States). 2nbdg, EVAD, and ISRIB were gained from Selleck chemistry (TX, United States). Antibody information is shown in Supplementary Table 1.