Anti cd44 bv650 clone im7

The following antibodies were used in a surface panel cocktail for the Bl-Eng2 peptide experiments: anti-CD45–Alexa Fluor 488 (clone 30-F11; Biolegend, catalog no. 103122), anti-major histocompatibility complex (MHC) class II tetramer–phycoerythrin (PE), anti-CD4–BUV395 (clone GK1.1; BD, catalog no. 563790), anti-CD8–peridinin chlorophyll protein (PerCP)-Cy5.5 (clone 53-6.7; Biolegend, catalog no. 100734), anti-CD44–BV650 (clone IM7; Biolegend, catalog no. 1033049), anti-CD11b–allophycocyanin (APC) (clone M1/70; Biolegend, catalog no. 101212), anti-CD11c–APC (clone N418; Biolegend, catalog no. 117310), anti-NK1.1–APC (clone PK136; Biolegend, catalog no. 108710), and anti-B220–APC (clone RA3-62B; Biolegend, catalog no. 103212).

T-cell epitopes were mapped within NSs antigen. IFN-γ ELISpot assays were performed with splenocytes from vaccinated mice by stimulation with a single peptide (15-mer; total 41 peptides). Peptides with high numbers of IFN-γ spots were identified, and the epitope sequence corresponding to high-affinity binding to mouse MHC class Ι, H-2L^d was identified using NetMHC 4.0 (Technical University of Denmark). The epitope peptide YPYLMAHYL was used to synthesize the H-2L^d dextramer (H-2L^d NSs_247–255 dextramer, Immunedex). Splenocytes of vaccinated mice were stained with PE–conjugated H-2L^d NSs_247–255 dextramer at 4 °C for 20 min, followed by washing with FACS buffer (PBS containing 1% FBS and 0.05% sodium azide). For live/dead and surface staining, the following reagent and antibodies were used: Live/Dead Fixable Red Dead Cell stain kit (Invitrogen), anti-CD19–PE-CF594 (clone 1D3, BD Biosciences, 562291, 1:100), anti-CD8–BV510 (clone 53-6.7, BD Biosciences, 560776, 1:100), anti-CD62L–BV605 (clone MEL-14, BD Biosciences, 563252, 1:50), anti-CD44–BV650 (clone IM7, BioLegend, 103049, 1:50), and anti-CD3–Alexa Fluor 700 (clone 500A2, eBioscience, 56-0033-82, 1:100).