Annexin 5 fitc propidium iodide apoptosis detection kit

HK-2 cells cell apoptosis was detected using an Annexin V-FITC/Propidium Iodide Apoptosis Detection Kit (Abcam, Cambridge, UK) following the manufacturer’s instructions. The cells were fixed in FACS fixing buffer containing 1% paraformaldehyde. Flow cytometry (FACS Calibre, BD Biosciences, NJ, USA) was used to perform the assay. The positive cells were analysed using the FlowJo software (Ashland, DE, USA).

Apoptotic cells were assessed using an annexin V-FITC/propidium iodide apoptosis detection kit (Abcam) via flow cytometry in MDA-MB-231 and MCF-7 cells (Cell Bank of Type Culture Collection of the Chinese Academy of Sciences). Since shRNA lentivirus vector expressing GFP will influence the FACS signal of Annexin-FITC, siRNA was used to knock down MIR4435-2HG in the flow cytometric analysis of apoptotic assays. Briefly, breast cancer cells (5×10⁴ cells/ml) transfected with 50 nM siMIR4435-2HG (5′-CCCAGAUUUAAGGGCUAUUTT-3′) or siNC (5′-UUCUCCGAACGUGUCACGUTT-3′) were seeded in 6-well culture plates. After 48 h, cells were digested by 0.25% EDTA-free trypsin, washed 2 times with cold PBS, resuspended in 100 µl binding buffer, and stained with Annexin V-FITC as well as propidium iodide (100 µg/ml). After 15 min incubation at 37°C in the dark, apoptotic cells were analyzed using a FACS Calibur flow cytometer (Becton, Dickinson and Company), and analyzed using Cell Quest software v.3.3 (Becton, Dickinson and Company). The experiment was performed in triplicate.

The apoptotic cells were measured by flow cytometry using an annexin V–FITC/propidium iodide apoptosis detection kit (Abcam, Cambridge, UK) in U2OS cells. The fluorescence intensity was detected at 488 nm using flow cytometry. Cells were sorted by the FACSCalibur flow cytometer (Becton Dickinson, San Diego,CA), and analyzed using CellQuest software (Becton Dickinson).