Tetramer-based Enrichment of Immune Cells

Tetramer enrichment was performed similarly as previously described^{40 (link), 46 (link)}. Single-cell suspensions were prepared from the spleens or thymi harvested from wild-type littermate and Sult2b1-deficient mice. Red blood cells (RBCs) were then lysed with ACK buffer. With equal numbers of total cells from each groups, PE-conjugated HY Uty-WMHHNMDLI:Db tetramers (MBLI) were added to the cell suspension at a 1:10 dilution. Cells were incubated for 1 h at 4 °C co-stained with anti-CD8. After washing, cells were resuspended in PBS, 2 mM EDTA and 0.5% BSA, and anti-PE microbeads (Miltenyi Biotec) were added to the cell suspension. Samples were incubated at 4 °C for 15 min and then washed. Cells were applied to pre-washed MACS LS columns. Following three washes, the bound fraction was eluted and resuspended in FACS buffer (PBS, 2 mM EDTA and 5% FBS). Tetramer-bound fractions were stained with 1:100 dilution of fluorophore-conjugated antibodies to the following markers: F4/80 (BM8), Gr-1 (RB6-8C5), CD11b (M1/70), CD11c (N418), Ter119 (TER-119), CD19 (6D5), CD4 (RM4-5) and Live/Dead Aqua amine (Life Technologies). After staining, the washed samples were collected on a BD LSR II and analyzed using FlowJo (Tree Star).