Rat and mouse serum

40–80 × 10⁶ frozen spleen cells were thawed into DMEM with 10% fetal calf serum and 100 U/mL penicillin plus 100 µg/ mL streptomycin. Cells were centrifuged and resuspended 50 µL of ice-cold fluorescence-activated cell sorting (FACS) buffer composed of 1× DPBS and 1% newborn calf serum (Thermo Fisher, cat#26010074). PostF PE/DyLight650 conjugated tetramers were added at a final concentration of 25 nM in the presence of 2% rat and mouse serum (Thermo Fisher) and incubated at room temperature for 10 min. S2P PE tetramers were then added at a final concentration of 5 nM and incubated on ice for 25 min, followed by a 10 mL wash with ice-cold FACS buffer. Next, 50 μL of anti-PE-conjugated microbeads (Miltenyi Biotec, Auburn, CA, USA, cat#130-090-855) were added and incubated on ice for 30 min, after which 3 mL of FACS buffer was added and the mixture was passed over a magnetized LS column (Miltenyi Biotec, cat#130-042-401). The column was washed once with 5 mL ice-cold FACS buffer and then removed from the magnetic field and 5 mL ice and 5 mL ice-cold FACS buffer was forced through the unmagnetized column twice using a plunger to expel cells retained in the column as the bound cell fraction.

1–2 × 10⁸ frozen PBMCs or 4–8 × 10⁷ frozen spleen cells were thawed into DMEM with 10% fetal calf serum and 100 U/mL penicillin plus 100 µg/mL streptomycin. Cells were centrifuged and resuspended in 50 µL of ice-cold fluorescence-activated cell sorting (FACS) buffer composed of phosphate-buffered saline (PBS) and 1% newborn calf serum (Thermo Fisher, cat#26010074). PostF APC/DyLight755 and PE/Dylight650 conjugated tetramers were added at a final concentration of 25 nM in the presence of 2% rat and mouse serum (Thermo Fisher) and incubated at room temperature for 10 min. PreF APC and PE tetramers were then added at a final concentration of 5 nM and incubated on ice for 25 min, followed by a 10 mL wash with ice-cold FACS buffer. Next, 50 μL each of anti-APC-conjugated (Miltenyi Biotec, cat#130-090-855) and anti-PE-conjugated (Miltenyi Biotec, cat#130-048-801) microbeads were added and incubated on ice for 30 min, after which 3 mL of FACS buffer was added and the mixture was passed over a magnetized LS column (Miltenyi Biotec, cat#130-042-401). The column was washed once with 5 mL ice-cold FACS buffer and then removed from the magnetic field and 5 mL ice-cold FACS buffer was pushed through the unmagnetized column twice using a plunger to elute the bound cell fraction.