Annexin 5 pi double staining

The MSCs from the experimental group and control group were harvested and washed with PBS; then the cells were labeled by Annexin V–PI double staining (KeyGen Biotech, Jiangsu, China). Ultimately, the samples were analyzed by flow cytometry. For Sal B administration, MSCs were cultured in a medium containing Sal B for 24 h before change to the different concentration of dexamethasone.

A549 cells were obtained from the Institute of Radiation Medicine, Peking Union Medical College (China), and cultured in RPMI‐1640 containing 10% fetal bovine serum (FBS) and antibiotics. The cells were incubated at 37°C in a humid environment with 5% CO₂. The above cell culture reagents were purchased from Gibco (Grand Island, USA). MPPa, Cell Counting Kit‐8, 2′,7′‐dichlorofluorescin diacetate and Hoechst 33342 were obtained from Sigma‐Aldrich. Annexin V/PI double staining and JC‐1 mitochondrial membrane potential detection kits were manufactured by Keygen Biotech (Nanjing, China).
Rabbit monoclonal antibodies against human caspase‐3 and ‐9, Bcl‐2, and Bax, respectively, were manufactured by Cell Signaling Technology (Danvers, MA). Anti‐β‐actin and anti‐cytochrome‐c primary antibodies as well as secondary antibodies were purchased from Abcam (Cambridge, UK). The PDT equipment was manufactured by Chongqing Jingyu Laser Technology Co. Ltd. (Chongqing, China).

Cells were treated with ART for 24 h prior to treatment with 2 or 6 Gy irradiation. Apoptosis was measured using propidium iodide (PI)/Annexin-V double staining following manufacturer’s instructions (Keygen Biotech, Nanjing, China). Cells were harvested 24 h after treatment with ART; apoptotic fractions were measured using flow cytometry (Beckman, USA). The Annexin-V+/PI- cells are early in the apoptotic process, the Annexin-V+/PI + cells indicating late apoptosis. The percentage of both kinds of cells was counted. The Annexin-V-/PI + cells are considered to be necrotic cells.
For tissue samples, 5 μm xenograft sections were deparaffinized in xylene and hydrated in decreasing concentrations of ethanol, and the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed following manufacturer’s instructions (Keygen Biotech, Nanjing, China). Ten random fields from 4 slides per group were examined. TUNEL-positive brown nuclei within tissues were counted. Data were expressed as the percentage of apoptotic cells per field.