Muse annexin 5 kit

The apoptotic impact of the radiated riboflavin on C6 cells was assessed using the Muse Annexin V kit of Merck Millipore. The C6 cell line was grown in 24 wells including plates; split into four groups: either not treated or treated with 31.25, 62.5 and 93.75 μM of radiated riboflavin. Following a 24‐h treatment period, the cell culture media was disposed, and the cell line was rinsed using PBS. Thereafter, the cell line was washed with EDTA‐containing PBS and maintained in trypsin–EDTA for 3–5 min. The detached cells were exposed to 10 min‐centrifugation at 150 g, and the pellet was then reconstituted with new medium. In the meantime, 100 μL of solution of annexin V plus propidium iodide (provided with the kit) were combined in an Eppendorf tube. The produced cell suspension was then introduced at a volume of 100 L into this mixture. These two solutions were incubated at room temperature for 20 min in the dark. Finally, the cells were analysed exploiting the Muse TM Cell Analyser (Merck Millipore, Hayward, CA, USA).

Apoptosis was measured by flow cytometry using a MUSE Annexin V kit (Merck, Germany). Cells were seeded at an initial concentration of 1 × 10⁶ cells/mL in the absence or presence of OAT-449 or vincristine. After 24 h, cells were collected, centrifuged (400 g, 4 °C, 5 min), and washed with 1 mL of PBS. Afterward, cells were incubated for 15 min in the dark on ice in 100 μL of the Annexin V incubation buffer (0.1 M HEPES, 25 mM CaCl₂, 1.4 M NaCl, pH 7.4), with 1 µL of Annexin V-FITC. After incubation, 400 μL of the Annexin binding buffer and 2 μL of PI (50 μg/mL) were added. Measurements were performed using a MUSE analyzer (Merck, Germany) and data were analyzed using MUSE Analysis 14.1 software.