Propium iodide

LPS was purchased from Sigma Aldrich (St Louis, MO, USA) and originated from S. Typhimurium (LPS1) and E. Coli (LPS2), respectively. All stimulations were performed for a total of 6 h except for rhS100A9 (20 h). IL-1β and HMGB1 was from R&D Systems. Recombinant human S100A9 (rhS100A9) was a gift from Active Biotech AB and a detailed description on endotoxin-free S100A9 generation and purification has been published previously [15 (link)] and was used in the presence of calcium and zinc (Ca²⁺ ≥200 μM; 10 μM ZnCl₂ [34 , 35 (link)]). Supernatants from stimulated or siRNA transfected cells were harvested and analyzed using human inflammatory cytokine cytometric bead array (CBA; BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions or using IL-6 and IL-8 Quantikine ELISA (R&D Systems, Minneapolis, MN, USA). Annexin V-allophycocyanin (APC) and propium iodide (PI) staining was performed according to the manufacturer’s instructions (BD Biosciences). The cycloheximide (CHX) experiments (Sigma Aldrich) where performed by adding 10 μg/ml CHX, with or without 100 ng/ml LPS for 6 h.

We detached 5 × 10⁵ cells from the plates using 3mM EDTA/PBS and resuspended in 500 μl of ice-cold PBS. Annexin V-fluorescein isothiocyanate (FITC) staining was performed according to the manufacturer’s protocol (BD Pharmingen, San Diego, CA, USA). For cell cycle profile analysis, cells were fixed using 80 % ethanol, stored overnight at 4 °C, and then spun at 1,500 rpm for 5 minutes at 4 °C using a centrifuge (Eppendorf 5810R). Pellets were washed with cold PBS + 1 % serum, mixed, spun for 5 minutes at 1,200 rpm, and stained with propium iodide (PI)/RNase solution (BD Pharmingen). All analysis was performed using FACS Diva software on a BD FACSCanto II flow cytometer.