Annexin 5 fluorescein isothiocyanate fitc early apoptosis detection kit

FACS (BD Cell Quest^® version 3.3) was carried out using an Annexin V-Fluorescein isothiocyanate (FITC) Early Apoptosis Detection Kit (Cell Signaling Technology, Inc.). This kit included Annexin V-FITC and propidium iodide (PI) to confirm changes in the apoptotic populations of eutopic endometrial cells from patients with and without endometriosis treated with formononetin. Eutopic endometrial cells from patients with and without endometriosis were cultured and treated with 0, 80 μM formononetin for further 24 h. The plate on which the cells adhered was rinsed with Dulbecco’s phosphate-buffered saline (DPBS), and cells were detached using trypsinization. The cells underwent resuspension in 1X binding buffer (BD Biosciences). An amount of 1 µL Annexin V-FITC and 12.5 µL PI were added to 96 µL cell suspension. After incubation for 15 min at 37 °C, each apoptotic cell population was analyzed by the BD Cell Quest^® version 3.3. software (Becton Dickinson and Company, Franklin Lakes, NJ, USA).

Flow Cytometry method was applied to measure the apoptotic cells with application of Annexin V-Fluorescein Isothiocyanate (FITC) Early Apoptosis Detection Kit (Cell Signaling Technology, USA) following instruction provided. Briefly, cells were trypsinized and then resuspended. Cell suspensions were incubated with Annexin V-FITC and propidium iodide (PI) for 10 min at room temperature (RT) and avoid light. Samples should be detected by flow cytometry (Beckman FC 500, USA) timely. Necrotic cells (Quadrant UL), late apoptotic cells (Quadrant UR) and early apoptotic cells (Quadrant LR) and were counted separately.