Annexin 5 pi staining

To observe nuclear condensation, 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich)-stained cells were observed using a fluorescence microscope (IX71; Olympus, Tokyo, Japan). Cell apoptosis levels were analyzed using nuclear propidium iodide (PI; Sigma-Aldrich) staining and flow cytometry (FACSCalibur; Becton Dickinson, San Jose, CA) with the excitation set at 488 nm and emission detected with the FL-2 channel (565-610 nm). The distribution of cells in the different phases of the cell cycle was calculated using MetaMorph software (Molecular Devices, Downingtown, PA, USA). Annexin V/PI staining was performed according to the manufacturer's instructions (eBioscience, San Diego, CA, USA). The cells were detected in the FL-1 (480–530 nm) and FL-2 channels (565–610 nm) using the FACS Calibur. For apoptosis analysis, the samples were analyzed using CellQuest Pro 4.0.2 software (Becton Dickinson), and quantification was performed using WinMDI 2.8 software (The Scripps Institute, La Jolla, CA, USA). Apoptosis levels are reported as the percentage (%) of cells in the sub-G₁ phase and in the gate of annexin V⁺ PI⁻ cells.

Cells were resuspended and fixed by adding 1 ml of ice-cold 70% ethanol to phosphate-buffered saline (PBS) and were then stored at 4°C. Before the analysis, the fixed cells were washed in PBS and incubated with propidium iodide (PI) staining solution [(0.04 mg/ml of PI (Sigma-Aldrich) and 0.1 mg/ml of RNase A (Sigma-Aldrich)] for 30 min at room temperature. The cells were analyzed using a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA, USA) with an excitation at 488 nm and emission in the FL-2 channel (565–610 nm). We analyzed ten thousand cells per sample. Small cellular debris was excluded by gating on a forward scatter plot, and the apoptotic cells were gated and quantified in the sub-G₁ phase. Annexin V/PI staining was performed according to the manufacturer's instructions (eBioscience, San Diego, CA, USA). The cells were detected in the FL-1 (480–530 nm) and FL-2 channels (565–610 nm) using the FACS Calibur.

Monocyte/macrophage THP1 cells are a gift from Dr. Amir Yazdi (Lausanne, Switzerland) and cultured in RMPI Medium 1640 (Gibco, Illkirch, France) with 10% fetal calf serum (Hyclone, Cramlington, UK) and penicillin/streptomycin (100 U/ml, Invitrogen). For experiments, THP1 were differentiated for 3 h with 0.5 μM PMA, washed and plated overnight (2 × 10⁵ cells/well). Cells were stimulated for indicated times, the supernatant was collected for immediate ATP measurement and/or stored for further IL-1β quantification.
Cell death was monitored by MTT using a standard protocol. Thiazollyl blue tetrazolium bromide (Sigma) solution was added onto the cells after supernatant collection and incubated for 2 h at 37 °C, and a 10% SDS acetic acid solution is then added. MTT reduction to formazan was quantified by an absorbance microplate reader (EL800, BioTek, Colmar, France) at 610nm (KC4 software). Apoptotic and necrotic cell death of primed THP1 cells was also monitored using AnnexinV/PI staining (eBioscience, San Diego, CA, USA). Cells were gently detached using repeated aspiration and expulsion of cold PBS. After centrifugation, cells were resuspended in annexin V binding buffer and stained for 20 min with Annexin V and propidium iodide. Data were collected on a BD FACSCanto and analysed using Flowjo software (Tree Star, OR, USA).