Annexin 5 fitc binding solution

bEnd.3 cells were X-irradiated 24 h after plating. The cells were collected at different times after irradiation. For NSCs, after co-culture with bEnd.3 cells for different times, NSCs were dispersed into single cells and collected. The collected cells were then resuspended in Annexin V-FITC binding solution and stained with Annexin V FITC/PI (BD Biosciences, USA) for 15 min at RT. Apoptotic cells were determined on a flow cytometer (BD FACSVerse, USA). ModFit LT3.2 Software was used for quantification of apoptosis.

Adherent HTR-8/SVneo cells were washed with PBS for three times and trypsinized. Cells were resuspended in 195 µl Annexin V-FITC binding solution (BD Biosciences) at a density of 5×10⁵/tube. Cells were subsequently mixed with 5 µl Annexin V-FITC and 10 µl PI staining solution (BD Biosciences) and incubated for 20 min at room temperature in the dark. Cell apoptotic rates were calculated by detecting the percentage of early and late apoptotic cells using flow cytometry (DxFLEX; Beckman Coulter, Inc.) and CytExpert software (version 2.0.0.283; Beckman Coulter, Inc.).

There were three experimental groups of CC cell lines for this analysis: i) Ctrl, siRNA negative control; ii) siRNA#1 transfection; and iii) siRNA#2 transfection. HeLa, C33A, SiHa and CaSki cells in the logarithmic growth phase were collected and added to 6-well plates, and then transfected with siRNA at 30-50% confluence. Cells were digested and the transient transfection was terminated after 72 h to create a single-cell suspension. Cells were centrifuged at 500 × g for 5 min, and washed twice with PBS. Then, 100 μl Annexin V-FITC binding solution (BD Biosciences) was added to re-suspend the cells. After adding 5 μl PE-Annexin V and 5 μl 7-AAD staining solution, cells were incubated at room temperature for 15 min in the dark. Then, 400 μl Annexin V-FITC was added and the cells were detected with a FC 500 flow cytometer (Beckman Coulter, Inc.). The results were analyzed using FlowJo v10 software (FlowJo LLC) to create a scatter plot. The living cells were on the lower left quadrant (FITC^-/7-AAD^-), early apoptotic cells on the lower right quadrant and late apoptotic and dead cells on the upper right quadrant.