Muse annexinv and dead cell assay kit

The EMT6 cells were seeded
at a density of 1.0 × 10⁵ cells/well in 35 mm Petri
dishes containing 2 mL of culture medium. Following 24 h of incubation,
the cells were divided into the following groups: control (no treatment);
laser (irradiated with a light dose of 15 J/cm²); Mal₃–TEG–Ce6 (treated with 0.8 M Mal₃–TEG–Ce6); and PDT (treated with 0.8 μM Mal₃–TEG–Ce6 and then irradiated with a light dose
of 1, 5, or 15 J/cm²). The cells were incubated with 0.8
μM Mal₃–TEG–Ce6 for 4 h. After washing
with fresh media, the cells were irradiated by a 671 nm laser light
(8.3 mW/cm²; 1, 5, and 15 J/cm²) emitted by
a DPPS laser (HangZhou NaKu Technology Co., Ltd., China, Zhejiang)
using an optical fiber with a microlens delivery attachment. Apoptosis
was assessed 4 h after the laser irradiation using the Muse Annexin
V and Dead Cell Assay Kit (Merk Millipore, Germany) according to the
manufacturer’s protocols. Annexin V was used to detect phosphatidylserine
on the external membrane of the apoptotic cells. The ROS generation
was assessed 4 h after laser irradiation using the Muse Oxidative
Stress Kit (Merk Millipore, Germany) according to the manufacturer’s
protocols; this kit determines the percentage of cells that are negative
(healthy cells) and positive for ROS (cells containing ROS). Single-cell
suspensions were then loaded onto the Muse Cell Analyzer (EMD Millipore
Co.).