Flow Cytometry

LECs were detached with EDTA-free trypsin and washed with phosphate buffer saline (PBS) twice. The cells were harvested (1-5×10⁵). The binding buffer (50 µL) was mixed with 7-amino-actinomycin D (7-ADD) solution, and the cells were added to the mixture, followed by incubation at room temperature in the dark for 5-15min. Flow cytometry was done by using annexin V-PE apoptosis detection kit (Invitrogen, Beijing, China) at the FL2 channel. At the excitation wavelength of 488 nm and the emission wavelength of 578 nm, orange fluorescence was observed. The 7-ADD was detected via the FL3 channel. Red fluorescence was observed at the excitation wavelength of 546 nm and emission wavelength of 647 nm. All groups were performed in triplicate.