Fcs express v4.0 research edition software

Samples were analyzed on a FACSCanto flow cytometer (Becton Dickinson, Mountain View, NJ, USA), which was calibrated daily. Cells were incubated with 10% FBS for 10 min on ice to prevent nonspecific binding. Afterwards, the cells were incubated with the antibodies or corresponding isotype controls in the concentration recommended by the manufacturer for 20 min on ice without light exposure. After washing the cells with PBS supplemented with 0.1% BSA, the samples were analyzed. Monocytes were gated using CD14 expression, forward scatter (FSC), and sideward scatter (SSC), and the data were analyzed applying FCS Express V4.0 research Edition software (De Novo Software, Glendale, California, USA).

A daily calibrated FACS-Canto flow cytometer (Becton Dickinson, Mountain View, CA, USA) was used to perform phenotypic analysis. To prevent nonspecific binding, cells were incubated with 10% fetal calf serum on ice for 10 min before staining with appropriate fluorophore coupled secondary antibodies, or isotype-specific immunoglobulin-labelled monoclonal antibodies for 20 min over ice in the dark. Monocytes were gated by forward (FSC), side scatter (SSC), and CD14 expression. For intracellular cytokine staining, monocytes were fixed in 2% paraformlaldehyde/PBS for 30 min at room temperature (RT) and washed three times with PBS. Afterwards monocytes were permeabilized utilizing a permeabilization buffer (purchased from Thermo Fisher Scientific, Hennef, Germany) according to the manufacturers’ recommendations. Data was analyzed using the FCS Express V4.0 research Edition software (DeNovo Software, Glendale, California, USA).