Fitc conjugated annexin 5 and propidium iodide

A propidium iodide (PI) and annexin V-FITC-flow cytometry assay (BD Pharmingen) was used to detect the apoptosis rate in the cells after the induction of different glucose concentrations. Briefly, 5×10⁴cells per well were cultured in 6-well plates in the complete culture medium for 24 hours. Then, the complete culture medium was aspirated and discarded from each well, cells were treated with medium containing different concentrations of glucose (5.5mM, 15mM and 25mM) for 24 hours and 48 hours. Adherent cells were detached with 0.25% trypsin without EDTA in 1×PBS. Cells were harvested in PBS and centrifuged at 2000 rpm for 5 minutes. Then the cells were washed in PBS, re-suspended in binding buffer (BD Pharmingen), and stained with FITC-conjugated annexin V and propidium iodide (Pharmingen, Becton Dickinson Co., San Diego, CA, USA). After staining, the cells were incubated for 15 minutes in the dark at room temperature. Cells were analysed by flow cytometry (FACS Calibar; Becton-Dickinson) using Cell Quest software.

Apoptosis was assessed by Annexin V/Propidium Iodide staining according to the manufacturers' instructions (BD Pharmingen). After 24 hours of treatment cells were labeled with FITC-conjugated Annexin V and Propidium Iodide (Becton-Dickinson, Italy). Fluorescence Activated Cell Sorting (FACS) analysis was performed using a FACS-Canto Cell Cytometer and the CellQuest software (Becton-Dickinson, Italy).

HT29 cells were exposed to different concentrations of deoxyshikonin (0, 25, 50 and 100 μg/mL) at a fixed time (24 or 48 h) in six-well culture plates (5 × 10⁵ cells per well). Then, we harvested the cells and supernatant, washed in PBS and resuspended 1 × 10⁶ cells/mL in 1× binding buffer. Next, FITC-conjugated Annexin V and propidium iodide (BD Biosciences Pharmingen, Franklin Lakes, NJ) were added to the 100 μL cell suspension. Finally, the samples were measured with a BD FACSCanto™ II (BD Biosciences, San Jose, CA) after incubation in the dark for 15 min at room temperature. Annexin V single positive represented the cells in the early apoptotic stage, while both Annexin V and PI positive represented the cells in the last stage of apoptosis, and PI single positive cells represented necrotic cells. The experiment was performed three times.