Fluo 3 am ester

To detect intracellular Ca²⁺, Fluo-3, which is known to dramatically increase fluorescence after calcium ions binding, was used. In accordance with the manufacturer’s protocol, human neutrophils were incubated with 5 μM Fluo-3 AM ester (ThermoScientific, USA) for 60 min at room temperature followed by washing with PBS. After loading, PMNLs were incubated according to the experimental protocol with or without additives in HBSS/HEPES medium. For kinetics assays, PMNLs were seeded in fibrinogen-coated 96-well plates (0.5 × 10⁶/mL HBSS/HEPES), then 1 µM oligonucleotides were added. Changes in Fluo-3-Ca-fluorescense intensities some time before and at least 20 min after ODN addition were recorded on a fluorometric plate reader upon excitation of 485 nm and emission of 538 nm for 20 min at 37 °C. Flow cytometry was also used. In this case, cells (1 × 10⁶/mL HBSS/HEPES medium) were incubated with 1 µM oligonucleotides for 20 min, with or without the addition of opsonized S. typhimurium (OS) for the last 10 min of incubation. At the end of the incubation, the samples were placed on ice followed by immediate acquisition on Cytomics FC 500 Flow Cytometry System. Fluorescence was collected by photomultipliers at 525 nm.

Intracellular Ca²⁺ ions were detected by staining with Fluo-3, which is known to dramatically increase fluorescence after binding of calcium ions, was used. According to the manufacturer’s protocol, human neutrophils were incubated with 5 μM Fluo-3 AM ester (Thermo Fisher Scientific, Waltham, MA, USA) for 60 min at room temperature, followed by washing in PBS. Cells were then seeded in fibrinogen-coated 96-well plates (1 × 10⁶/mL of HBSS/HEPES) and incubated at 37 °C in 5% CO₂. Changes in fluorescence intensity upon excitation at 485 nm and emission at 538 nm were monitored at CLARIOstar fluorescence microplate reader for at least 60 min after stimulus injection, followed by data analysis using MARS Data Analysis Software, version 3.30 (both hardware and software are from BMG Labtech, Ortenberg, Germany).