Multiparameter flow cytometry analysis

Single cell suspensions were stained with a viability dye, followed by incubation with purified rat anti-mouse CD16/CD32 (mouse BD Fc Block, clone 2.4G2) as previously described (22 (link)). Cells were then stained for 25 min at 4°C in the dark using antibodies targeting the following markers: CD69 (H1.2F3, Biolegend), CD4 (RM4-5, BD Biosciences), CD11a (2D7, BD Biosciences), and CD44 (IM7, Tonbo), (MEL-14, Biolegend). Cells were then washed and prepared for flow cytometry analysis. Data was acquired using a BD LSR-II instrument, configured with 488 (blue), 633 (red), 407 (violet), and 532 (green)-nm lasers. Data were analyzed using Flowjo software (FlowJo, LLC), version 10.