Foxp3 gfp cre

Spleen and thymus of six-week-old NOD.Foxp3-GFP/cre (Stock No: 008694, Jackson Laboratory) were processed using frosted glass slides and passed through a 40-micron filter to create single cell suspensions. Red blood cells (RBC) were lysed with ammonium-chloride-potassium buffer prior to staining for flow cytometry. Samples were stained with Fixable Live/Dead Near IR (Invitrogen), washed once with stain buffer (PBS + 2% FBS + 0.05% NaN₃), and Fc receptors were blocked with anti-CD16/32 for 5 minutes at 4°C (Clone 2.4G2, BD Biosciences). Samples were stained for 30 minutes at 4°C with the following anti-mouse antibodies: CD3e-Brilliant Violet (BV) 605 (145-2C11, BioLegend), CD4-PerCP/Cy5.5 (RM4-5, Thermo Fisher Scientific), CD8a-BV711 (53-6.7, BioLegend), CD44-PE-Cy7 (IM7, BioLegend), CD62L-APC (MEL-14, BioLegend), and CD221-PE (3B7, Santa Cruz Biotechnology). Samples were washed once with stain buffer prior to data acquisition on an LSRFortessa (BD Biosciences) and analysis with FlowJo (v10.6.1; Tree Star).

The University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee (OUHSC IACUC) approved all mouse procedures described in this study and all mouse study methods were carried out in accordance with the relevant guidelines approved by the OUHSC IACUC. The C57BL/6J, FoxP3-GFP-Cre, A2Ar^−/−, CCR6^−/−, and CCR7^−/− mice used in this study were purchased from Jackson Laboratories.