Apc anti mouse h 2kb

2×10⁶ spleenocytes in 800 µL of 10% RPMI-1640 culture media were incubated with 200 µL of SIINFEKL, polyK-SIINFEKL, SIINFEKL-polyK, or SIINFEKL-AHX-polyK peptide dilution (0.19 μg/μL). Alternatively, 7.9×10⁹ vp mixed with 37.5 µg of polyK-SIINFEKL (OVA-PeptiCRAd) in 200 µL of 10% RPMI-1640 was applied. The PeptiCRAd complex was prepared as described previously. After 2 h of incubation cells were washed and stained with either APC anti-mouse H-2K^b bound to SIINFEKL or APC Mouse IgG1, κ Isotype Ctrl (BioLegend, San Diego, CA, USA), and the samples were analyzed by flow cytometry.

RMA-S cells were cultured until logarithmic phase. Cells were washed twice in PBS and incubated in serum-free RPMI medium for 90 minutes in 37°C. Following incubation, 2^∗10⁵ cells/well were incubated with 0.1-100 μM peptide in 37°C. 18 hours later, surface H2-Db and H2-Kb levels were assessed by FACS via APC anti-mouse H-2Kb and PE anti-mouse H-2Db antibodies (BioLegend), respectively. Analysis was done using FlowJo software (Tree Star). All peptides were purchased from GenScript.