Annexin 5 fitc pi staining kit

HK-2 cells were seeded, transfected and treated with LPS in 6-well plates as previously mentioned, but LPS treatment was 6 h to observe apoptosis which was measured using an annexin V-FITC/ PI staining kit and flow cytometry (Becton Dickinson, San Jose, CA). After incubation, cells were washed twice with PBS and cell density was set at 1 × 10⁶/mL with precooling Hank’s Balanced Salt Solution (HBSS). Then cells were incubated with fluorescein-conjugated annexin V and PI in the dark for 15 min at room temperature. Stained cells (1 × 10⁵ cells/sample) were analyzed by flow cytometry and apoptosis was quantified.

The apoptosis of OCI-LY7 cells was tested using an Annexin V-FITC/propidium iodide (PI) staining assay. OCI-LY7 cells were cultured in 12-well plates and transfected with pre-miR-10a, anti-miR-10a, BCL6 siRNA, or the BCL6 overexpression plasmid to induce apoptosis. Pre-miR-control, anti-miR-control, control siRNA, and control plasmid served as negative controls. Cells were cultured overnight with serum-depleted medium, and then the cells were harvested. We detect the OCI-LY7 cells apoptosis of under normal or serum deprivation over night (Fig. S5). Flow cytometry analysis of apoptotic cells was performed using an Annexin V-FITC/PI staining kit (BD Biosciences, CA, USA). After washing with cold PBS, the cells were resuspended in binding buffer (100 mM HEPES at pH 7.4, 100 mmol/L NaCl, and 25 mmol/L CaCl₂), followed by staining with Annexin V-FITC/PI at room temperature in the dark for 15 min. Apoptotic cells were then evaluated by gating PI- and Annexin V-positive cells using a fluorescence-activated cell-sorting (FACS) flow cytometer (BD Biosciences, San Jose, CA).

First, the apoptotic potential of individual formulation was determined using Annexin V-FITC/PI staining kit (BD Biosciences, NJ). Briefly, MCF-7 and MDA-MB-231 breast cancer cells (2 × 10⁵) were seeded in a six-well plate and incubated for 24 h. The cells were then treated with respective formulations for 24 h. Subsequently, the cells were then washed, collected, and stained with Annexin V-FITC (2 µL) and PI (2 µL) for 15 min. The cells were then analyzed using a FACS Calibur^TM instrument. Cells that were Annexin-V⁺ were early apoptotic; Annexin-V^–/PI^– were live cells; whereas Annexin-V⁺/PI⁺ cells were late apoptotic or necrotic.
For cell cycle analysis, cells (2 × 10⁵) were seeded in a six-well plate and incubated for 24 h. The cells were then treated with respective formulations and incubated for 24 h. The cells were collected by trypsinization and fixed with 70% cold ethanol for 1 h. Subsequently, the cells were incubated with 10 µL PI (10 mg/mL) and 5 µL ribonuclease (10 mg/mL) for 30 min at 37 °C in the dark. The cell cycle analysis was performed on the FACS Calibur flow cytometer (BD Biosciences, NJ) (10,000 cells per measurement).