Annexin 5 pe apoptosis detection kit

Apoptosis and cell death assay was performed using Annexin V Apoptosis Detection Kit PE (eBioscience, catalog: 88-8102-74) as previously described (27 (link)), and cell death was monitored using a flow cytometer (BD FACS Fortessa).

Annexin-V staining was performed to differentiate apoptosis from necrotic cell death induced by ASNPs. Annexin-V protein has a high affinity to phosphotidyl serine, which is translocated from the inner to the outer leaflet of the plasma membrane at early-stage of apoptosis. The conjugation of Annexin-V with the fluorescent probe PE facilitates measurements by flow cytometric analysis. The use of 7AAD staining helps to distinguish between apoptosis and necrosis owing to differences in permeability of 7AAD through the cell membranes of living and damaged cells. Cell number, concentrations, and culture conditions were similar to those used in the cell cycle analysis. Treated cells were harvested and washed twice in PBS. The staining was carried out following the manufacturer’s instruction (eBioscience Annexin V Apoptosis Detection Kit PE, Catalog Number: 88–8102). Cells were incubated in the dark for 15 min at room temperature in 200 µl binding buffer containing Annexin V-PE (10 µl) and 7AAD (5 µl). After incubation, 300 µl binding buffer was added to each sample, and cells were kept on the ice. The 488-nm argon ion laser was used for excitation with BD FACS Calibur (Becton Dikinson, San Jose, CA, USA) flow cytometry system. PE was detected in FL-2, while 7AAD was detected in FL-3. 10000 cells were acquired and data analyses were performed using the Flowjo version 7.6.1.

BM, spleens, and peritoneal cells were obtained from mice. After staining with specific antibodies, cells were analyzed using a FACS Aria III or a FACS Canto II (BD Bioscience) and then by FlowJo software (Treestar Inc.). For sorting of B cell lineages, BM cells were stained with PE/Cy5-conjugated antibodies against CD3, CD4, CD8, Gr-1, NK1.1, and TER119. After washing, cells were incubated with anti-Cy5 microbeads (Miltenyi Biotec) and B cells were isolated by negative isolation with Auto MACS (Miltenyi Biotec). BM B cells were then stained with specific antibodies, and specific cell populations were isolated using a FACS Aria III (BD Bioscience). For counting cell numbers in the BM and spleen, CountBrightTM absolute counting beads (Invitrogen) were used. Apoptotic cells were detected using an Annexin V Apoptosis Detection Kit PE (Thermo Fisher Scientific). For RNA-sequencing analysis, CD11b⁺F4/80⁺ peritoneal macrophages were sorted from the peritoneal cavity of naive mice and CD11b⁻ B cells and M-B cells were sorted from the BM 48 h after LPS treatment.

Cell apoptosis was determined using the Annexin V Apoptosis Detection Kit PE (Thermo Scientific, Waltham, USA) according to the manufacturer’s instructions. Briefly, 6 × 10⁵ cells were harvested and resuspended in 500 μL of 1 × binding buffer. After adding 5 μL Annexin V-PE and 10 μL 7-aminoactinomycin D (7-AAD) to each tube, the cells were incubated in the dark at room temperature for 10 min. Then, the samples were analyzed using flow cytometry (BD Biosciences, San Jose, CA, USA).

Annexin V PE Apoptosis Detection Kit (Cat number: 88-8102, eBioscience, USA) was used to label apoptotic cells according to manufacturer's instructions. LNCaP and DU145 cells with or without rCCL18 stimulation were seeded at a density of 5 × 10⁵ cells in 6-well plates. Annexin V-PE was added 48 hours later and apoptotic cells were identified with a flow cytometer according to manufacturer's protocol. Data were expressed as of three independent experiments.