Benzidine staining

A total of 1×10⁶ cells were collected into a 1.5-ml EP tube with 500 µl PBS, following which 15 µl 0.4% benzidine solution and 10 µl 3% H₂O₂ were added and the suspension was incubated for 3 min at room temperature. Then, 1 µl 5% sodium nitroprusside solution was added. Then, 10 min later at room temperature, 10 µl suspension was collected for observation (magnification, ×200) using an inverted microscope (Olympus Corporation). The positive cells were blue-black in color, and 200 cells were counted to calculate the positive cell rate.