Cells were planted in six-well plates and divided into four groups as described above. We then administered 100 μL/mL nimotuzumab for 24 hours and the cells received 6 Gy irradiation the next day. The cells were harvested 48 hours later and fixed overnight with 70% ethanol. They were then resuspended with PBS containing 100 μg/mL RNAase and 50 μg/mL propidium iodide (PI). A flow cytometer (FACScan; BD, San Jose, CA, USA) was used to test the DNA content and cycle distribution. The quantification of cells in different phases was analyzed using CELLQuest (BD, San Jose, CA, USA).
As for the apoptosis assay, 48 hours after irradiation the cells were collected, resuspended with 500 μL binding buffer, and stained with 5 μL Annexin-V-EGFP and 5 μL PI according to the operation manual (Annexin-V-EGFP/PI apoptosis kit; Beyotime, Nanjing, Jiangsu, People’s Republic of China). Cells were then turned to the flow cytometer for the apoptosis test.
As for the apoptosis assay, 48 hours after irradiation the cells were collected, resuspended with 500 μL binding buffer, and stained with 5 μL Annexin-V-EGFP and 5 μL PI according to the operation manual (Annexin-V-EGFP/PI apoptosis kit; Beyotime, Nanjing, Jiangsu, People’s Republic of China). Cells were then turned to the flow cytometer for the apoptosis test.