Tacs annexin 5 fitc kit

Measurements of treatment-induced apoptosis were performed using the TACS ® Annexin V-FITC Kit (R&D Systems). Cells were plated at low density in 6-well plates and allowed to grow for five days under the indicated treatment. Detached cells were collected and placed in a collection tube. Attached cells were lifted using TrypLE ™ (Gibco) for 15 minutes at 37°C. Collected cells were centrifuged at 500xg for five minutes, washed with 1x ice-cold phosphate-buffered saline (PBS), and incubated for 15 minutes at room temperature in 100 μL of staining solution (1:200 Annexin-FITC, 1x binding buffer (R&D Systems), 1:10 propidium iodide (PI) in H 2 O). Cells were diluted 1:4 in 1x binding buffer and 10-30,000 cellular events were analyzed via flow cytometry (Cytoflex S, Beckman Coulter). To differentiate cells from debris, FSC-A (x) and SSC-A (y) gating was performed using Cytobank (RRID:SCR_014043). Finally, apoptotic cells were defined as cells positively labelled for Annexin-FITC staining and further defined as either PI-positive or -negative.

Cellular apoptosis was detected according to the protocol of R&D TACS ® Annexin V-FITC Kit, containing identified Annexin V-FITC and propidium iodide (PI) in binding buffer. Annexin V binds to the phosphatidylserine (PS) in the early apoptosis, while PI is a marker of late apoptotic and necrotic cells. First, cells were seeded and incubated with 16 µM Andro, 200 nM DDP, or both. After 48 h, the cells were harvested by trypsinization, washed twice with cold phosphate-buffered saline (PBS), and diluted to 1 × 10 6 cells/mL in 1× binding buffer. Then, 5 µL of Annexin V and PI solution was added to every 100 µL of cell suspension (about 1 × 10 5 cells). Cells were gently mixed and incubated at room temperature (RT) in the dark for 15 min. Each tube was added with 400 µL of 1× binding buffer. Finally, cells were sorted by flow cytometry on FACScan (Becton Dickinson, Franklin Lake, NJ, USA) within 1 h and then analyzed by FCS Express 4 software.