Flow cytometry diva software

Cultured cells were incubated for 20 min at 4 °C with appropriate antibodies APC-conjugated anti-BDCA-4, FITC-anti-CD123 (Miltenyi, Bergisch Gladbach, Germany), PE.cy7-anti-CXCR4 clone 12G5 (Biolegend, San Diego, CA), Live/Dead Green Kit (ThermoFisher Scientific) or with appropriate isotype-matched control antibodies (5μg/mL each) in PBS containing 2% mouse serum (Sigma, Saint Louis, MO) and FC-receptor blockers (BD Biosciences, San Jose, CA). For IRF-7 intracellular staining, cells were fixed with 2% PFA then permeabilized with 0.5% saponin before being stained for 30 minutes at 4 °C with anti-IRF-7 antibody (BD Biosciences). Flow cytometry analysis was performed on a flow cytometry Canto II flow cytometer using flow cytometry Diva software (BD Biosciences, San Jose, CA). FlowJo software (Treestar, Ashland, OR) was used to analyze data.

Cultured cells were incubated for 20 min at 4 °C with the following antibodies mouse IgG1 PE-conjugated TRAIL clone RIK-2 (1/100) (BD Bioscience, San Jose, CA), mouse IgG1 APC-conjugated BDCA-4 clone REA380 (1/100), mouse IgG2a FITC-CD123 clone AC145 (1/100) (Miltenyi, Bergisch Gladbach, Germany), mouse IgG2a FITC-HLADR clone L243 (1/200), mouse IgG2a PercP-cy5.5-CCR7 clone G043H7 (1/50), mouse IgG1 APC-CD40 clone 5C3 (1/50), mouse IgG1 BV421-CD80 clone 2D10 (1/50), mouse IgG2b AF488-CD86 clone IT2.2 (1/50), mouse IgG1 PE-CXCR4 clone 12G5 (1/100) (Biolegend, San Diego, CA) or with appropriate isotype-matched control antibodies (5 μg ml⁻¹ each) in PBS containing 2% foetal bovine serum (Sigma, Saint Louis, MO) and FC-receptor blockers (BD Biosciences, San Jose, CA). Flow cytometry analysis was performed on a BD Canto II or LSR II flow cytometer using flow cytometry Diva software (BD Biosciences, San Jose, CA). FlowJo software (Treestar, Ashland, OR) was used to analyse data.