Pe conjugated streptavidin

For proliferation studies, cells were labelled with 10 μM proliferation dye eFlour 670 (eBiosciences, #65-0840-90) for 10 min on ice. Labelling was stopped by adding 4-5 volumes of complete culture medium for 5 min on ice. Cells were then subjected to flow cytometry analysis (day0) or maintained in culture for additional 5 days prior to analysis by Flow Cytometry. For lectin staining, Peanut Agglutinin (PNA, #B-1075), Sambucus Nigra Lectin (SNA, #B-1305), Aleuria Aurantia Lectin (AAL, #B-1395) and Maackia Amurensis Lectin II (MAA, # B-1265-1), all from vector laboratories, were used. Cells were blocked in carbon-free blocking buffer (vector laboratories, #SP-5040) for 20 min on ice, and then incubated with biotinylated lectins diluted in PBS containing 5% carbon-free blocking buffer. Lectins were then detected with FITC-conjugated streptavidin (Biolegend, #405201), Alexa Flour 647-conjugated streptavidin (Biolegend, #2068269) or PE-conjugated Streptavidin (Biolegend, #410504) in PBS for 20 min at 4°C. To differentiate between live and dead cells, all samples were stained with DAPI (Sigma, #32670, dilution 1:1000) prior to data acquisition. Data were acquired using an LSR II flow cytometer (BD) and analysed using FlowJo version 10.8.0.

Mouse splenocytes were cultured in a complete RPMI-1640 medium (GenDEPOT, Katy, TX, USA) supplemented with fetal bovine serum (10%, Hyclone, South Logan, UT, USA) and 1% penicillin/streptomycin (Welgene, Gyeongsan, Korea). Anti-mouse TCR-β-PE, CD45.1-Percp Cy5.5, CD3-APC Cy7, CD8-FITC, CD44-PE, and IFN-γ-FITC antibodies as well as CFSE cell proliferation tracing dye were purchased from Tonbo Bioscience (San Diego, CA, USA). Anti-mouse TNF-α-PE Cy7, APC-conjugated streptavidin, and PE-conjugated streptavidin were purchased from Biolegend (San Diego, CA, USA). Gp^33-41 class I pMHC tetramer was provided by the NIH Tetramer Core Facility (Atlanta, GA, USA).