Foxp3 fix and perm kit

Mouse spleens were isolated and strained through a 70‐μM strainer to disperse cells. Red blood cells were lysed using RBC lysis buffer (Invitrogen, Waltham, MD). Non‐specific antibody binding sites were blocked by incubation with the CD16/32 Fc‐receptor blocking antibody (BioLegend, San Diego, CA; cat. no. 101201, RRID:AB‐312800). For analyses of cell surface markers, cells were labelled with fluorescent‐conjugated antibodies in phosphate‐buffered saline containing 2% bovine serum albumin. For FOXP3 staining, cells were labelled with cell surface markers, then fixed using the FoxP3 fix and perm kit (eBioscience, Waltham, MA), and then incubated with APC‐Foxp3 antibody (ThermoFisher Scientific, Waltham, MA; cat. no. 17‐4776‐41, RRID:AB_1603281). Flow cytometry measurements were performed with the Canto II instrument (BD Scientific, San Jose, CA; Canto II Flow Cytometer, RRID:SCR_018056) using the flow cytometry core (https://pathology.wustl.edu/research/core‐facilities/flow‐cytometry‐fluorescence‐activated‐cell‐sorting/). FACS data were analysed with FlowJo software (FlowJo, Ashland, OR; RRID:SCR_008520).