Flow Cytometry of Mouse Cells

Cells were pre-incubated in PBS containing TruStain fcX™ Fc Receptor Blocking Solution (BioLegend) diluted 1:500, and eBioscience™ Fixable Viability Dye eFluor™ 780 diluted 1:1000 in order to block non-spiecific binding of immunoglobulins to the Fc receptors and exclude dead cells, respectively. Subsequently, cells were stained with specific anti-mouse antibodies for 45 min at 4°C. For intracellular staining of cytokines, the cells were fixated in BD Cytofix™ Fixation Buffer (BD Biosciences) prior to staining and 0.5% saponin (Sigma-Aldrich) was included in the flow cytometry buffer. After staining, cells were analyzed on FACSCanto II flow cytometer (BD Biosciences) and the data were analyzed and processed using FlowJo software.