Foxp3 buffer kit

Blood samples were collected by retro-orbital bleed. Tissues were harvested after isoflurane overdose followed by cardiac puncture. Tetramer and flow cytometry staining and ex vivo peptide stimulation for intracellular cytokine staining were performed as described (Smithey et al., 2011 (link)). For thymocyte gating, non-αβ T-cell precursors were excluded with a dump gate that included the following cell surface markers: γδ TCR, NK1.1, CD19, and B220. Samples were fixed and permeabilized using the Foxp3 buffer kit (eBioscience, San Diego, CA, USA) followed by intracellular staining. Samples were collected on a custom LSR Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using flowjo software (Tree Star, Ashland, OR, USA). All antibodies were purchased from eBioscience, BD, and Invitrogen. D^b-NS4b tetramer was provided by the NIH tetramer core facility (Emory University, Atlanta, GA, USA).

Antibodies used for flow cytometry analysis were: V500-CD3 (clone UCHT1, BD), APC-H7-CD8 (clone SK1, BD), V450-CD4 (clone RPA-T4, BD), PerCy5.5-CD4 (clone SK3), PE-CD25 (clone 2A3, BD), FITC-CD127 (clone HIL-7R-M21, BD), APC-CD39 (clone B249211, BioLegend), PE-Cy7-CD73 (clone AD2, BD), IgG1-APC (clone MOPC-21, BD), IgG1-PerCP-Cy5.5 (clone X40, BD), IgG1-PE-Cy7 (clone X40, BD), AF647-Foxp3 (clone 206D, Biolegend). Intranuclear FoxP3 staining was conducted using eBiosciences FoxP3 buffer kit (San Diego, CA, USA) according to the manufacturers' protocol. The LIVE/DEAD™ Fixable Violet dead cell stain kit (L34955, Invitrogen) was used to exclude dead cells. Stained cells were acquired using the BD FACSCanto II and analyzed using FlowJo software 10.4.2. Treg were defined as CD25⁺CD127^low/neg or CD4⁺FoxP3⁺ events.